Application of thiephine in preparation of medicine for treating premature ovarian failure

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of thienphine in preparation of a medicine for treating premature ovarian failure. The thiephine is used in combination with the MSC. The thiephine protects endogenous opioid peptides by inhibiting NEP activity, reduces follicular atresia and improves the ovarian microenvironment. The invention analyzes and studies that Thiorphan (NPE inhibitor) can inhibit NEP activity, protect endogenous opioid peptide (such as enkephalin), reduce follicular atresia and improve ovarian microenvironment, MSC can promote follicular survival and angiogenesis through paracrine (such as VEGF and IGF-1) and immunoregulation (such as inhibition of TNF-alpha and IL-6), and the combination of Thiorphan and MSC can achieve the effect of inhibiting tumor cells through dual mechanisms of drug protection and cell repair. A new thought is provided for treating premature ovarian failure.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to the application of thiophene in the preparation of a drug for treating premature ovarian failure. Background Art

[0002] Premature ovarian failure (POF) is an ovarian dysfunction disorder characterized by amenorrhea, ovarian atrophy, and decreased fertility in women before the age of 40. It is a difficult-to-treat type of female infertility. Conventional treatments for POF currently include hormone replacement therapy (HRT), combined biocorticosteroids, and dehydroepiandrosterone therapy. HRT only alleviates the symptoms of low estrogen levels and has no substantial effect on improving ovarian reproductive function. Furthermore, long-term HRT use can increase the risk of endometrial and ovarian cancer.

[0003] Multiple animal models and clinical trials have demonstrated that mesenchymal stem cell (MSC) transplantation can improve ovarian function through multiple pathways, including reducing granulosa cell (GC) apoptosis and promoting angiogenesis. Long et al. performed umbilical cord MSC transplantation on 61 patients with POF and found a significant increase in the number of antral follicles, dominant follicles, and mature follicles after treatment. Khadijeh et al. treated POF mice with MSC and found that MSC transplantation significantly restored the mice's hormone secretion capacity, reduced follicle apoptosis, promoted GC proliferation, and promoted the recovery of damaged ovarian tissue structure and function, saving fertility. They also found that the MSC-transplanted mice produced offspring. The mechanisms of MSC treatment for POF include: direct differentiation of MSC into oocytes; differentiation of MSC into GC; restoration of ovarian function through paracrine pathways; and regulation of ovarian immune function.

[0004] Although MSC transplantation is a promising treatment for POF, cell senescence directly affects in vitro cell expansion and leads to low therapeutic efficiency after MSC transplantation. Therefore, analyzing the molecular mechanism of MSC senescence and intervening to delay MSC senescence are of great significance to breaking the bottleneck of MSC treatment. Summary of the Invention

[0005] The purpose of the embodiments of the present invention is to provide the use of thiophene in the preparation of a drug for treating premature ovarian failure, aiming to solve the problems raised in the above background technology.

[0006] The embodiment of the present invention is achieved by using thiophene in the preparation of a drug for treating premature ovarian failure.

[0007] Preferably, the thiophene is used in combination with MSCs.

[0008] Preferably, the thiophene protects endogenous opioid peptides by inhibiting NEP activity, reducing follicular atresia, and improving the ovarian microenvironment.

[0009] Another object of an embodiment of the present invention is to provide a drug for treating premature ovarian failure, wherein the drug includes thiophene.

[0010] Preferably, the drug further comprises MSCs.

[0011] Preferably, the drug is an injectable preparation, and the thiophene and MSC are injected simultaneously.

[0012] The examples of the present invention analyze and study that Thiorphan (NPE inhibitor) can inhibit NEP activity, protect endogenous opioid peptides (such as enkephalin), reduce follicular atresia, and improve the ovarian microenvironment, while MSCs can promote follicle survival and angiogenesis through paracrine (such as VEGF, IGF-1) and immune regulation (such as inhibition of TNF-α, IL-6). The combination of Thiorphan and MSCs can provide a new approach for the treatment of premature ovarian failure through the dual mechanism of "drug protection + cell repair". BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 NEP expression in MSCs is upregulated in the RS model provided in the embodiments of the present invention (A. RT-PCR results; B. western blot results; C. immunofluorescence results);

[0014] Figure 2 GO analysis of the signaling pathways and differentially expressed genes affected by NEP knockdown in MSC cells provided in the embodiment of the present invention;

[0015] Figure 3 The results of the Thi pretreatment provided in the embodiment of the present invention can inhibit MSC aging (A. western blot detection of PTEN / p-PTEN and AKT / p-AKT (A); B. expression of P16 and P27; C. β-Gal staining results);

[0016] Figure 4 Results of the present invention's embodiment of knocking down NEP and high concentrations of Thi inhibiting cell cycle and proliferation and promoting cell apoptosis (A. shRNA transfected cells to knock down NEP; B. WST-1 assay for cell proliferation; C. flow cytometry for cell cycle; D&E. western blot assay for cell cycle and apoptosis-related protein expression);

[0017] Figure 5 The results of ELISA detection of FSH, LH and E2 expression in POF mice provided in the embodiment of the present invention;

[0018] Figure 6 The ovarian weight and HE staining results of POF mice provided in the embodiments of the present invention;

[0019] Figure 7The results of the detection of the expression levels of the two genes mRNA of VEGF and Rad50 in the ovarian tissues of normal mice and POF mice provided in the embodiments of the present invention;

[0020] Figure 8 Schematic diagram of Thi and MSC treatment of POF mice provided in an embodiment of the present invention;

[0021] Figure 9 The present invention provides the statistical results of FSH, LH, and E2 expression in mice after POF treatment, as well as ovarian wet weight and number of offspring born. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0024] Example 1: Molecular mechanism of Thiorphan delaying MSC aging by inhibiting NEP:

[0025] (1) NEP expression in senescent MSC cells is significantly upregulated:

[0026] Obtain mouse inguinal fat tissue, remove blood vessels and cut into 2-3 mm pieces. 3 To prevent the destruction of the microenvironment for MSC growth, collagenase digestion is not required. Small pieces of tissue are attached to a 10 cm culture dish, ensuring that the fat section is adhered to the wall downwards. A small amount of culture medium (2-3 ml) is added and cultured continuously for 5-7 days in a special mesenchymal stem cell culture medium (STEM CELL Co.) to wait for AMSC to grow out of the tissue. During this period, fresh culture medium is slowly replaced. The volume of culture medium is gradually increased as AMSC grows. MSC is cultured to G15 (total culture 55 days) by continuous passage method to prepare RS model and detect the expression of MSC in RS model, such as Figure 1 As shown, RT-PCR ( Figure 1 A) and western blot results ( Figure 1 B) showed that NEP expression in senescent cells was significantly upregulated, and immunofluorescence also showed that NEP expression in senescent MSC cells was higher than that in the control group ( Figure 1 C);

[0027] (2) NEP promotes MSC aging by regulating the PTEN-AKT signaling pathway:

[0028] By combining transcriptome sequencing with GO analysis, Figure 2 As shown in the results, it was found that after NEP knockdown, 26 signaling pathways in the cells were significantly affected, among which the PI3K-Akt signaling pathway and Ras signaling pathway had the most changes in gene expression;

[0029] like Figure 3 As shown in the results, the PTEN-AKT signaling pathway is an important signaling pathway that regulates cell proliferation, senescence, and apoptosis. The expression of PTEN / p-PTEN and AKT / p-AKT was detected in RS senescent cells cultured for 30 days. It was found that the expression of p-PTEN was significantly upregulated at 20 days and 30 days, while the expression of p-AKT was significantly decreased ( Figure 3 A), when the NEP inhibitor Thiorphan (0.5 mM, Thi) was added to the culture system, the expression of p-PTEN was significantly inhibited, while the expression of p-AKT was significantly upregulated compared with the control group at 20 and 30 days of culture ( Figure 3 A); In addition, the expression of P16 and P27 in MSCs also increased significantly at 20 and 30 days of culture ( Figure 3 B), while after adding 0.5 mM Thi, the expression of P16 was significantly inhibited on the 20th day of culture, and the expression of P27 was significantly inhibited on both the 20th and 30th days of culture ( Figure 3 B); The results of cell senescence staining also showed that adding 0.5mM Thiamin to the culture system can effectively inhibit MSC senescence ( Figure 3 C);

[0030] (3) Knockdown of NEP and high concentration of Thi inhibit cell cycle and proliferation and promote cell apoptosis:

[0031] like Figure 4 As shown, MSCs were transfected with NEP shRNA ( Figure 4 A), and treated cells with high concentration of Thi (2 mM), detected cell proliferation by WST-1, and found that knocking down NEP or inhibiting NEP activity inhibited cell proliferation ( Figure 4 B) and induce G2 / M arrest ( Figure 4 C);

[0032] Western blot results also showed that the expression of cell cycle proteins and kinases, including cyclin B1, cyclin D1, CDK1, CDK2 and CDK4, was inhibited when cells were transfected with shNEP and treated with 2 mM Thi. Figure 4 D); At the same time, the expression levels of apoptosis proteins and cell cycle regulatory proteins also changed, including caspase 3, caspase 7 and P21 activation ( Figure 4 E).

[0033] In summary, the mechanism by which NEP affects cell aging is as follows: high levels of NEP can induce cell aging by activating PTEN, inhibiting the AKT signaling pathway; low concentrations of Thi (0.5 mM) can delay cell aging by inhibiting the PTEN signaling pathway; at the same time, NEP is a protein essential for cell survival. If it is knocked down or high concentrations of Thi (2 mM) are used, it will inhibit cell proliferation, induce cell cycle arrest, and induce cell apoptosis.

[0034] Example 2: Establishment of POF model in mice:

[0035] Eight-week-old female BALB / c mice were anesthetized with sodium pentobarbital and injected intraperitoneally with cis-platinum at a dose of 3 mg / kg body weight once a week for 4 weeks. A control group was injected with an equal dose of normal saline in parallel. After 4 weeks, blood was collected from the orbits and the serum was separated by centrifugation. The expression levels of hormones FSH, LH, and E2 were detected by ELISA. Figure 5 As shown in the results, the expression levels of FSH and LH were significantly upregulated, while the expression level of E2 was significantly downregulated. The ovaries of mice were observed and the ovaries of mice treated with cis-platinum were reduced in size and weight ( Figure 6 A), ovarian tissue was taken for HE staining to observe the number and quality of follicles. It was found that the number of follicles in the cis-platinum group was significantly reduced ( Figure 6 B).

[0036] Example 3: Establishment of a database of genes driving premature ovarian failure by NGS transcriptome sequencing:

[0037] Ovarian tissues from normal mice and POF mice were collected and ground in liquid nitrogen to extract RNA. cDNA libraries were constructed using an RNA-seq library preparation kit (Illumina TruSeq RNALibrary Prep Kit) and sent to a sequencing company for next-generation transcriptome sequencing (Illumina HiSeq).

[0038] The sequencing results were compared between the CT group and the POF group, and screening was performed according to lgFC>1.5. 20 genes with relatively high FPKM values ​​and down-regulated genes related to development, aging, cell proliferation, and apoptosis were obtained, as shown in Table 1:

[0039] Table 1

[0040]

[0041]

[0042] Among them, VEGF and Rad50 genes were detected by RT-PCR. Figure 7As shown in the figure, the expression of VEGF and Rad50 genes was significantly downregulated in POF mice.

[0043] Example 4: Thiorphan promotes MSC treatment of POF by inhibiting NEP overexpression:

[0044] POF mice were prepared according to the method in Example 2 and treated starting at week 4. The mice were divided into the following groups, with 6 mice in each group:

[0045] (1) PBS group served as control;

[0046] (2) MSCs were injected into the tail vein at a concentration of 1×10^6 cells / 100 μl at the starting point of 4, 5, 6, and 7 weeks;

[0047] (3) MSCs treated with Thi for 7 days were injected into the tail vein at a cell concentration of 1×10^6 cells / 100 μl. The injection time points were the starting point of 4, 5, 6, and 7 weeks.

[0048] (4) injection of Thi alone at a concentration of 5 mg / kg, with the injection time points being the starting point of 4, 5, 6, and 7 weeks;

[0049] (5) Inject MSCs into the tail vein and simultaneously inject 5 mg / kg Thi at the starting point of 4, 5, 6, and 7 weeks;

[0050] After the 8th week, blood was collected from the eyeballs and the expression of FSH, LH and E2 was detected by ELISA. Three mice were sacrificed and the ovarian tissues were taken and weighed. RNA was extracted from the ovarian tissues and the expression of VEGF and Rad50 was detected by RT-PCR. The other three mice were cohabited with the male mice for 7 days and mated. After another 20-25 days, the number of female pregnancies and litters was counted ( Figure 8 ), the result is as follows Figure 9 As shown in Figure 2, among the four treatment groups, the treatment effect of simultaneous injection of MSC and Thi was the best, and the expression of FSH and LH was significantly reduced by ELISA ( Figure 9 A and B), while E2 expression was significantly upregulated ( Figure 9 C); After 4 weeks of treatment, the ovarian weight also showed that the ovarian weight of the MSC+Thi treatment group recovered most significantly ( Figure 9 D); After POF treatment, mating was performed again, and the statistics of the number of offspring born also showed that the number of offspring born in the MSC+Thi treatment group was significantly higher than that in the other treatment groups.

[0051] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. The application of thiophene in the preparation of drugs for the treatment of premature ovarian failure.

2. The use of thiophene according to claim 1 in preparing a drug for treating premature ovarian failure, characterized in that: The thiophene is used in combination with MSCs.

3. The use of thiophene according to claim 2 in preparing a drug for treating premature ovarian failure, characterized in that: The thiophene protects endogenous opioid peptides by inhibiting NEP activity, reducing follicular atresia, and improving the ovarian microenvironment.

4. A drug for treating premature ovarian failure, characterized in that: The drugs include thiophene.

5. The drug for treating premature ovarian failure according to claim 4, characterized in that The drug also includes MSCs.

6. The drug for treating premature ovarian failure according to claim 4, characterized in that The medicine is an injection-type preparation, and the thiophene and MSC are injected simultaneously.

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