Application of Compound C in inhibiting sheep uterine contraction

Abstract

The invention belongs to the technical field of biology, and discloses an application of Compound C in inhibition of uterine contraction of sheep, the Compound C significantly reduces expression of autophagy related genes LC3, BECN1, ATG5 and ATG7 by regulating an AMPK / mTOR signal channel, further inhibits autophagy of sheep uterine smooth muscle cells, and realizes targeted inhibition of uterine contraction. The optimal concentration of the Compound C in the preparation is 10 <-6 > mol / L, the administration dosage is 0.01 mg / kg of sheep body weight, the administration mode is intramuscular injection, and the Compound C can be combined with oxytocin to reverse the uterine contraction promoting effect of oxytocin. Cell experiments and animal experiments prove that the Compound C can improve uterine smooth muscle cell ultramicrostructure disorder, reduce autophagosome formation, delay the recovery speed of uterine horn of postpartum sheep, effectively inhibit abnormal uterine contraction and provide a new strategy for pregnancy maintenance and abortion reduction, and the action is milder.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of Compound C in inhibiting uterine contractions in sheep. Background Technology

[0002] Uterine smooth muscle contractions can induce normal parturition and rapid uterine involution in pregnant animals, but uterine contractions during pregnancy can lead to abortion. Three key physiological processes for successful pregnancy maintenance include maintaining uterine resting state, stable placental function, and maternal-fetal immune balance. Factors affecting uterine resting state include the maternal endocrine environment, the contractile activity of uterine smooth muscle, autophagy levels, and external stress stimuli. Maintaining uterine myometrium at rest and inhibiting premature contractions are the physiological basis for prolonging pregnancy. Abnormal activation and excessive autophagy of uterine smooth muscle are key limiting factors leading to pregnancy failure and decreased reproductive efficiency.

[0003] Autophagy, a key mechanism in uterine smooth muscle cell metabolism, is closely related to smooth muscle contraction function. Studies have shown that autophagy activity in the myometrium is significantly enhanced during the onset of labor, clearing dysfunctional mitochondria through mitophagy, optimizing ATP supply, and providing energy for uterine contractions; however, autophagy dysregulation may also lead to abnormal uterine contractions.

[0004] Existing uterine contraction inhibitors (such as β2-adrenergic receptor agonists) mostly target the contraction signaling pathway directly, aiming to forcibly inhibit the activated contraction pathway. Although they can temporarily suppress uterine contractions, they are prone to causing side effects such as cardiovascular problems in ewes. Compound C (Dorsomorphin) is a pyrazolopyrimidine compound and a specific inhibitor of adenosine monophosphate-activated protein kinase (AMPK). AMPK is a key sensor of cellular energy metabolism, and its inhibition can affect the downstream mammalian target of rapamycin (mTOR) signaling pathway, thereby regulating autophagy. Compound C has been shown to inhibit autophagy in research fields such as tumors and myocardial ischemia, but its function and application in regulating uterine contractions in ruminants have not yet been systematically reported. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes the application of Compound C in inhibiting uterine contractions in sheep. By providing a concentration of Compound C that inhibits autophagy of uterine smooth muscle cells and uterine muscle contractions, this invention suppresses the upstream mechanism of autophagy, providing a new strategy for achieving mild and targeted inhibition of uterine contractions.

[0006] To achieve the above objectives, the present invention provides the application of Compound C in inhibiting uterine contractions in sheep.

[0007] Preferably, Compound C inhibits uterine contractions by suppressing autophagy in sheep uterine smooth muscle cells.

[0008] Preferably, Compound C inhibits uterine smooth muscle cell autophagy by regulating the AMPK / mTOR signaling pathway and reducing the expression of autophagy-related genes LC3, BECN1, ATG5, and ATG7.

[0009] The application of Compound C in the preparation of an inhibitory agent for sheep uterine contractions is also provided, with a concentration of Compound C of 10. -8 -10 -4 mol / L.

[0010] Preferably, the optimal concentration of Compound C is 10. -6 mol / L.

[0011] Preferably, the dosage of Compound C is 0.01 mg / kg of sheep body weight, and the administration method is intramuscular injection.

[0012] Preferably, the formulation is used in combination with oxytocin to inhibit the contractile effect of oxytocin on the sheep uterus.

[0013] Preferably, the preparation method of Compound C is as follows: dissolving Compound C in DMSO to prepare a 10... -3 The concentrated stock solution (mol / L) should be stored in the dark at -80°C and diluted with PBS before use.

[0014] Compared with the prior art, the present invention has the following advantages and technical effects: 1) This invention is the first to discover and confirm that Compound C can effectively reverse postpartum uterine contractions in sheep caused by oxytocin, and has a clear effect on inhibiting uterine contractions in ewes, providing a novel target for uterine contraction regulation.

[0015] 2) This invention provides a reagent for inhibiting uterine smooth muscle contraction in sheep and its effective dosage. Experiments have confirmed that Compound C significantly reduces the mRNA expression levels of key autophagy-related genes (LC3, BECN1, ATG5, ATG7) by regulating the AMPK / mTOR autophagy signaling pathway. This inhibits uterine contractions from the upstream mechanism of abnormal uterine contractions, reducing the non-specific effects of traditional drugs that directly inhibit contraction pathways. The effect is more precise and gentle. Furthermore, Compound C 10 - 6 The effect is most significant at a concentration of mol / L.

[0016] 3) According to the present invention, injecting Compound C at a dose of 0.01 mg / kg body weight can significantly delay the recovery rate of uterine horns in ewes treated with oxytocin (OT). Specifically, the uterine recovery curve of the Compound C treatment group was not significantly different from that of the negative control group, but significantly different from that of the OT positive control group. This indicates that Compound C can effectively inhibit the uterine contraction-promoting effect of OT at the animal level.

[0017] 4) This invention provides a novel strategy with a novel mechanism of action, strong targeting and fewer side effects for inhibiting uterine contractions, maintaining pregnancy and uterine homeostasis, and provides important theoretical basis and application reference for further improving animal reproductive efficiency.

[0018] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0019] Figure 1 Ultrastructural images of uterine smooth muscle cells before and after Compound C treatment are shown under electron microscopy. In the figure, A represents the control group, B represents the OT treatment group, and C represents the OT+Compound C treatment group. Figure 2 The figure shows the expression levels of autophagy-related genes under different concentrations of compound C. In the figure, A represents the AMPK gene, B represents the mTOR gene, C represents the BECN1 gene, D represents the ATG5 gene, E represents the ATG7 gene, and F represents the LC3 gene. Different letters indicate that there is a significant difference between the two groups (P<0.05). Figure 3 Ultrasound images of the uterine horn of single lambing Hu sheep at 0-35 days postpartum in the control group (Control), OT treatment group (OT), and OT+Compound C treatment group (OT+Compound C); Figure 4 The study investigated the changes in the uterine horn diameter in single-lambed Hu sheep from 0 to 40 days postpartum. Control represents the control group, OT represents the OT treatment group, and OT+Compound C represents the OT+Compound C treatment group. Different letters indicate significant differences between the two groups (P<0.05). Figure 5 Ultrasound images of the uterine horn of twin-lambing Hu sheep from the control group (Control), OT treatment group (OT), and OT+Compound C treatment group (OT+Compound C) at 0-35 days postpartum; Figure 6The study investigated the changes in the cross-sectional diameter of the uterine horn in twin-lambing Hu sheep from 0 to 40 days postpartum. In this study, Control represents the control group, OT represents the OT treatment group, and OT+Compound C represents the OT+Compound C treatment group. Different letters indicate significant differences between the two groups (P<0.05). Detailed Implementation

[0020] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0021] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0022] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0023] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0024] In the examples, Compound C was purchased from MedChemexpress Biotechnology, Inc. (USA); DMSO was purchased from Thermo Fisher Scientific, Inc. (USA); and all reagents used in the qRT-PCR reaction system were from an RT-qPCR kit, which was purchased from Takara Corporation, Japan.

[0025] Example 1 Preparation of Compound C.

[0026] Accurately weigh 0.005g of Compound C and dissolve it in 12.55ml of DMSO to prepare a solution of 10... -3 The concentrated stock solution is dispensed in 100 μL portions and stored frozen at -80°C in the dark (dilute with PBS to the required concentration before use).

[0027] Example 2 The effect of Compound C on autophagy in sheep uterine smooth muscle cells.

[0028] The main instruments included a carbon dioxide cell culture incubator, a stereo microscope, and a transmission electron microscope (purchased from Thermo Fisher Scientific, USA).

[0029] 1) Collection of uterine smooth muscle cells: After collecting smooth muscle tissue from the lower segment of the uterus of a Hu sheep, the medical scissors were first sterilized by high temperature. Then, the uterine smooth muscle strip was carefully cut above the uterine incision with the scissors, and cut into pieces of about 0.5cm. 3 Tissue blocks (0.5×0.5×0.5cm) were prepared. The tissue blocks were rinsed with physiological saline to remove surface blood. After cleaning, the tissue blocks were transferred to centrifuge tubes, culture medium was added, the tubes were sealed, and the tubes were placed in an ice box to keep them warm. Within 5 hours, the tubes were transferred to the cell culture room for primary cell processing, cell purification, and passage culture.

[0030] 2) Sample collection and fixation: The cultured third-generation uterine smooth muscle cells were divided into three groups: control group, OT treatment group (OT (oxytocin) treatment for 30 min), and OT+Compound C treatment group (OT+10 min). -6 Treatment with Compound C at mol / L for 30 min, where the OT concentration was 10 mol / L. -6 mol / L. Discard the cell culture medium and gently rinse the cultured uterine smooth muscle cell smears (planted on Thermanox plastic coverslips) three times with pre-cooled 0.1M phosphate buffer (pH 7.4); immediately add pre-cooled 2.5% glutaraldehyde electron microscopy fixative (prepared with 0.1M phosphate buffer) and initially fix at 4°C for at least 2 hours; discard the fixative and thoroughly rinse the cell smears three times with 0.1M phosphate buffer for 15 minutes each time; then add 1% osmium tetroxide solution (prepared with 0.1M phosphate buffer) and fix at 4°C in the dark for 1.5 hours; after fixation, rinse again with 0.1M phosphate buffer three times for 15 minutes each time.

[0031] 3) Dehydration and permeation embedding: The fixed cell slides were sequentially placed in 50% ethanol → 70% ethanol → 90% ethanol → 100% ethanol (twice), each treatment lasting 10-15 min; then the ethanol was replaced with propylene oxide (or acetone) twice, each time for 15 min; the cell slides were then transferred to a propylene oxide:epoxy resin (Epon812) (1:1) mixture for permeation at room temperature for 2 h, and then transferred to pure epoxy resin for permeation at room temperature overnight; the fully permeated cell slides were placed in an embedding plate with the cell side facing up, fresh resin was injected, and polymerization was carried out at 60°C for 48 h.

[0032] 4) Preparation and staining of ultrathin sections: First, the embedded blocks were trimmed and cut into 1μm semi-thin sections. After toluidine blue staining, the target cell region was identified under an optical microscope. Next, ultrathin sections of 60-80nm were cut from the trimmed blocks and retrieved using a 150-mesh copper mesh covered with a Formvar membrane. The copper mesh was then immersed in a 2% uranium acetate-saturated ethanol solution and stained in the dark for 15 minutes. After rinsing with double-distilled water, it was immersed in lead citrate staining solution and stained in the dark for 8 minutes. After thorough rinsing with double-distilled water, it was air-dried. Images were then observed and acquired using a transmission electron microscope. The results are shown below. Figure 1 As shown.

[0033] The results showed that by observing uterine smooth muscle cells in the control group, the OT treatment group, and the OT+Compound C treatment group, the control group ( Figure 1 (As shown in Figure A) The mitochondria of the cells are normal in morphology, the membranes are not dissolved, the inner cristae are clear and not broken, and no edema is observed in the mitochondria. Figure 1 Cells treated with OT (as indicated by the green arrow in section A); Figure 1 (As shown in B) Ultrastructural disorder, mitochondrial swelling, and dissolution of the inner cristae ( Figure 1 As shown by the green arrow in section B), a large number of autophagosomes can be seen. Figure 1 (As shown by the red arrow in B); after processing by OT+Compound C ( Figure 1 As shown in C), the degree of disorder in the cell's ultrastructure was reduced compared to before, and the mitochondrial cristae were clearly visible. Figure 1 (As shown by the green arrow in C), a small number of autophagosomes ( Figure 1 (As indicated by the red arrow in C). This demonstrates that Compound C of the present invention can effectively inhibit autophagy in uterine smooth muscle cells.

[0034] Example 3 The effect of Compound C on the expression of autophagy-related genes.

[0035] 10 uterine smooth muscle cells -6 Pretreatment with mol / L OT. Three Compound C concentration gradients of 10 mol / L were set. -4 mol / L, 10 -6 mol / L, 10 -8 mol / L, the pretreated cells were randomly divided into the following four groups: PBS control group, 10 mol / L ... -4 mol / L Compound C treatment group, 10 -6 mol / L Compound C treatment group, 10 -8 Treatment group with mol / L Compound C. At 10 - 6After treatment with mol / L OT, different concentrations of Compound C were added, and the cells were placed back into the incubator for 1-2 hours to ensure that the inhibitors were fully effective. PBS was used in the same dose.

[0036] Based on sheep gene sequences in the NCBI database, primers were designed using Primer 5.0 software for the genes GAPDH, AMPK, mTOR, LC3, BECN1, ATG5, and ATG7 (primer sequences are shown in Table 1). GAPDH was used as an internal reference gene. The expression levels of key pathway genes and autophagy-related genes mRNA in the uterine smooth muscle cell control and treatment groups were detected. The relative expression levels of mRNA were calculated. The qRT-PCR reaction system used in the experiment is shown in Table 2, and the qRT-PCR reaction time is shown in Table 3.

[0037] Table 1 qRT-PCR primer sequences

[0038] Table 2 qRT-PCR reaction system

[0039] Table 3 qRT-PCR reaction time

[0040] The results are as follows Figure 2 As shown, treating sheep uterine smooth muscle cells with culture medium containing Compound C significantly reduced the expression of autophagy-related genes (LC3, BECN1, ATG5, ATG7) and decreased autophagy in uterine smooth muscle cells by regulating the AMPK / mTOR signaling pathway. The optimal concentration of Compound C was 10... -6 mol / L.

[0041] Example 4 The effect of Compound C on postpartum uterine contraction in sheep.

[0042] Forty-eight experimental Hu sheep were divided into two groups based on the number of lambs born: a single-lamb Hu sheep group (n=24) and a twin-lamb Hu sheep group (n=24). Different drug treatments were administered within 12 hours postpartum. The single-lamb Hu sheep group was further divided into a control group (3 ml 0.9% sodium chloride, n=8), an OT group (10 IU OT, n=8), and an OT+Compound C group (0.01 mg / kg sheep body weight Compound C + 10 IU OT, n=8). All treatments were administered intramuscularly. The twin-lamb Hu sheep group followed the same subgrouping method. The monitoring indicator was the change in uterine horn diameter during the recovery period (0-40 days).

[0043] The results are as follows Figure 3-4 As shown, the uterine recovery speed of ewes that gave birth to single lambs in the OT+Compound C group was not significantly different from the control group, but significantly different from the OT group. Figure 5-6 As shown, the uterine recovery rate in twin-lambing ewes in the OT+Compound C group was not significantly different from the control group, but significantly different from the OT group. This indicates that Compound C treatment reduced the uterine recovery rate after OT treatment and, to some extent, reversed the pro-contraction effect of OT on the uterus in Hu sheep.

[0044] In summary, this invention reveals for the first time the application of Compound C in inhibiting uterine contractions in sheep, clarifying its core mechanism of action through regulating the AMPK / mTOR signaling pathway, reducing the expression of autophagy-related genes, and inhibiting autophagy in uterine smooth muscle cells. Cellular experiments verified the protective effect of Compound C on the ultrastructure of uterine smooth muscle cells and its inhibitory effect on autophagy. Animal experiments confirmed its reversal effect on oxytocin-induced uterine contractions in sheep of different litter sizes. This invention determined the effective concentration range, optimal concentration, dosage, and administration method of Compound C, providing key technical parameters for its formulation development. Furthermore, this formulation can be used in combination with oxytocin, making it applicable in flexible scenarios. Compared with existing technologies, this invention has a clear target, mild side effects, and stable and reliable efficacy, providing a novel technical strategy for solving the problems of abortion and decreased reproductive efficiency caused by abnormal uterine contractions during sheep pregnancy. It also lays the foundation for research and formulation development related to the regulation of uterine contractions in ruminants, possessing significant theoretical and practical application value.

[0045] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of Compound C in inhibiting uterine contractions in sheep.

2. The application according to claim 1, characterized in that, Compound C inhibits uterine contractions by suppressing autophagy in sheep uterine smooth muscle cells.

3. The application according to claim 2, characterized in that, Compound C inhibits uterine smooth muscle cell autophagy by regulating the AMPK / mTOR signaling pathway and reducing the expression of autophagy-related genes LC3, BECN1, ATG5, and ATG7.

4. The application of Compound C in the preparation of an inhibitory agent for sheep uterine contractions, characterized in that the concentration of Compound C is 10... -8 -10 -4 mol / L.

5. The application according to claim 4, characterized in that, The optimal concentration of Compound C is 10. -6 mol / L.

6. The application according to claim 4, characterized in that, The dosage of Compound C is 0.01 mg / kg of sheep body weight, administered via intramuscular injection.

7. The application according to claim 4, characterized in that, The formulation is used in combination with oxytocin to inhibit the contractile effect of oxytocin on the sheep uterus.

8. The application according to claim 4, characterized in that, The preparation method of Compound C is as follows: Compound C is dissolved in DMSO to prepare a 10 -3 The concentrated stock solution (mol / L) should be stored in the dark at -80°C and diluted with PBS before use.

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