Use of ingenol HEP14 in preparation of drug, dosage form of HEP14-based drug, and preparation method for dosage form

Abstract

A novel medical use of ingenol HEP14, a dosage form of an HEP14-based drug, and a preparation method for the dosage form. The dosage form of the HEP14-based drug is an HEP14 / PLGA microsphere sustained-release dosage form formed by encapsulating the HEP14-based drug with a polylactic acid-glycolic acid copolymer, and has the functions of high HEP14 drug loading rate, high encapsulation rate, and long-acting HEP14 sustained-release. It has been found for the first time that HEP14 can promote production and autophagy of mitochondria in cells and enhance the function of mitochondria in cells, promote the secretion of antioxidant enzymes and remove reactive oxygen species, promote the growth and viability of ovarian follicle granulosa cells and restore the growth and viability of aging granules, promote the secretion of angiogenic factors, reduce the fibrosis of ovarian tissues, promote the angiogenesis of ovarian tissues, and promote the regeneration of aged ovarian follicles and ovarian endocrine functions, and thus has a good effect of treating and / or preventing related diseases caused by mitochondrial dysfunction, especially the effect of treating age-related ovarian decline.

Description

Application of ingenol HEPI4 in preparation of medicine, medicine dosage form and preparation method thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to a new medical use of ingenol HEPI4 (5beta-O-angeloyl-20-deoxyingenol, 5-O-angeloyl-20-deoxyingenol), a new medicine dosage form of HEPI4 and a preparation method thereof. BACKGROUND

[0002] Senescence refers to a process that organs and tissues of a living body gradually decline in function with age. Ovary is the earliest organ of female reproductive system to undergo senescence. Ovarian function decline is also an accelerator of whole body senescence. Ovarian insufficiency is caused by follicular development disorder and functional disorder of ovary due to reduction or depletion of follicle pool reserve in ovary, including premature ovarian failure (i.e., ovarian function decline before 40 years old, mainly caused by ovarian insufficiency due to ovarian damage after chemotherapy or / and radiotherapy) and age-related ovarian function decline, i.e., old ovarian senescence, which is a natural law, i.e., normal ovarian function gradually declines with age and leads to ovarian function depletion, resulting in female menopausal syndrome and complications such as osteoporosis, cardiovascular disease and neurodegenerative disease, which seriously endanger the physical and mental health of women. At present, there is no effective treatment for ovarian insufficiency at home and abroad. The most commonly used hormone replacement therapy for treating ovarian insufficiency can only alleviate the symptoms of low estrogen level, and has great side effects, and has no substantial therapeutic effect on improving ovarian reproductive function. Therefore, it is urgent to find / explore a more effective method for treating ovarian insufficiency in the field, which has been one of the most concerned research and development hotspots in the world.

[0003] Mitochondria are the organelles that produce reactive oxygen species (ROS) and are also the targets of ROS. During aging, the production and accumulation of ROS in oxidative metabolism continuously wear and consume mitochondria, leading to mitochondrial damage and functional decline to loss of function. Damaged mitochondria promote the accumulation of ROS, and this vicious cycle is considered the root cause of body aging and an important reason for the decline of female ovarian function. Mitophagy, as an important metabolic process in cells, mainly involves the cell wrapping damaged, aged or dysfunctional mitochondria through the autophagy system and degrading and recycling them through lysosomes to maintain the normal function of mitochondria in cells. Therefore, mitophagy is essential for cell survival and function. HEP14 is a 5β-O-angeloyl-20-deoxypulchinenol compound extracted from the whole grass of Euphorbia peplus Linn. and is a new activator of the protein kinase C (PKC) pathway. Currently, in addition to the effect of regulating lysosome generation that has been reported, other pathways of HEP14 have not been reported. In the process of exploring the therapeutic effect of HEP14 and its potential mechanism, in order to overcome the poor water solubility of HEP14 and obtain sustained and stable release of HEP14 in the circulation in vivo, we used poly(lactic-co-glycolic acid) (PLGA) as a drug carrier to prepare HEP14-loaded PLGA microspheres, called HEP14 / PLGA microspheres. PLGA is a biodegradable copolymer composed of polylactic acid and polyglycolic acid, which is widely used as a carrier to deliver therapeutic drugs due to its safety, manageable biodegradation rate and excellent drug loading performance. By adjusting the monomer ratio of lactic acid and glycolic acid, the degradation time of PLGA can be changed, which makes it particularly important in the field of biomedicine. PLGA is a drug carrier approved by the US Food and Drug Administration (FDA).

[0004] SUMMARY

[0005] In view of the fact that there is no effective treatment for ovarian dysfunction at home and abroad, and the conventional hormone replacement therapy has poor treatment effect and large side effects, the present application provides a new medical use of HEP14, and for the first time finds that the non-hormone natural small molecule compound HEP14 can activate the cell PKC-ERK1 / 2 signal pathway, promote the generation of cell mitochondria, autophagy and strengthen the function of cell mitochondria; promote the secretion of antioxidant enzymes and the clearance of active oxygen; promote the growth activity of ovarian follicular granulosa cells, restore the growth activity of aging granules; promote the secretion of angiogenic factors, reduce the fibrosis of ovarian tissue and promote the angiogenesis of ovarian tissue; promote the regeneration of old ovarian follicles and the endocrine function of the ovary, and therefore has good effects of treating and / or preventing diseases related to mitochondrial dysfunction, especially the effects of treating the decline of the ovary in old age. In addition, the present application also provides a dosage form of HEP14 drug, which is a HEP14 / PLGA microsphere sustained-release dosage form formed by wrapping HEP14 with poly(lactic-co-glycolic acid) (PLGA), and has high HEP14 drug loading rate, encapsulation rate and long-acting HEP14 sustained-release function. When the microsphere sustained-release dosage form is used for in vivo treatment, not only the poor water solubility of HEP14 is solved, but also the toxic side effects are small, so that HEP14 can stably exert the therapeutic effect in the body for a long time, and the subject in need does not need to be administered multiple times, thereby improving the compliance of the subject in need.

[0006] In the present application, the HEP14 is a compound with the structural formula as shown in the formula.

[0007] The present application solves the technical problems by adopting the following technical solutions:

[0008] In one aspect of the present application, the application of HEP14 in the preparation of a drug for activating PKC protein is provided.

[0009] In another aspect of the present application, the application of HEP14 in the preparation of a drug for inducing and activating ERK1 / 2 is provided.

[0010] In another aspect of the present application, the application of HEP14 in the preparation of a drug for promoting the generation of mitochondria is provided.

[0011] In another aspect of the present application, the application of HEP14 in the preparation of a drug for promoting the autophagy and function of mitochondria is provided.

[0012] In another aspect of the present application, the application of HEP14 in the preparation of a drug for promoting the secretion of antioxidant enzymes and the clearance of active oxygen is provided.

[0013] In another aspect of the present application, the application of HEP14 in the preparation of an anti-tissue fibrosis drug is provided.

[0014] Another aspect of the present invention provides the use of HEP14 in the preparation of drugs that promote tissue angiogenesis.

[0015] Preferably, the tissue is ovarian tissue.

[0016] In another aspect, the present invention provides the use of HEP14 in the preparation of a drug that promotes the growth of ovarian follicular granulosa cells.

[0017] In another aspect, the present invention provides the use of HEP14 in the preparation of a drug that promotes the secretion of hormones from ovarian follicular granulosa cells.

[0018] Another aspect of the present invention provides the use of HEP14 in the preparation of medicaments for the treatment and / or prevention of diseases related to mitochondrial dysfunction.

[0019] Another aspect of the present invention provides the use of HEP14 in the preparation of a medicament for treating ovarian dysfunction in the elderly.

[0020] In another aspect, the present invention provides the use of HEP14 in the preparation of a drug that promotes the regeneration of ovarian follicular cells.

[0021] Another aspect of the present invention provides the use of HEP14 in the preparation of a drug for restoring ovarian endocrine function.

[0022] The present invention also provides a drug formulation of HEP14, wherein the formulation is a HEP14 / PLGA microsphere sustained-release formulation formed by encapsulating HEP14 with polylactic acid-glycolic acid copolymer (PLGA).

[0023] Preferably, the HEP14 / PLGA microspheres have a smooth surface. A smooth surface facilitates the uniform, stable, and prolonged sustained release of HEP14, thus better exerting the therapeutic effect of HEP14 in vivo.

[0024] Preferably, the particle size of the HEP14 / PLGA microspheres is 0.0265 to 0.2373 micrometers.

[0025] More preferably, the particle size of the HEP14 / PLGA microspheres is 0.1003 to 0.2373 micrometers.

[0026] Preferably, the HEP14 loading rate in the HEP14 / PLGA microspheres is 2.1% to 3.1%.

[0027] Preferably, the encapsulation efficiency of HEP14 in the HEP14 / PLGA microspheres is 18.04% to 26.02%.

[0028] Another aspect of the present invention provides a method for preparing a drug dosage form of HEP14, comprising the following steps:

[0029] In a dark environment, polylactic acid-glycolic acid copolymer and HEP14 were dissolved in dichloromethane at a mass ratio of 5 to 10:1, and stirred with a magnetic stirrer at a speed of 400 to 500 rpm. Then, the resulting solution was added to an aqueous solution containing 1 to 2% (w / v) polyvinyl alcohol, and stirred with a magnetic stirrer at a speed of 300 to 500 rpm at 25°C to allow the dichloromethane to evaporate for at least 12 hours. Then, the microspheres were collected by centrifugation and repeatedly washed with distilled water to completely remove residual polyvinyl alcohol. Finally, the microspheres were placed in a freeze dryer for freeze drying to obtain the HEP14 / PLGA microsphere sustained-release formulation formed by polylactic acid-glycolic acid copolymer encapsulating HEP14.

[0030] Preferably, the polylactic acid-hydroxyacetic acid copolymer and HEP14 are dissolved in dichloromethane at a mass ratio of 5:1.

[0031] Compared with existing technologies, the present invention has the following beneficial effects: The present invention is the first to discover that the non-hormonal natural small molecule compound HEP14 can activate the cellular PKC-ERK1 / 2 signaling pathway, promote mitochondrial generation, autophagy, and enhance mitochondrial function; promote the secretion of antioxidant enzymes and scavenge reactive oxygen species; promote the growth vitality of ovarian follicle granulosa cells and restore the growth vitality of senescent granulosa cells; promote the secretion of angiogenesis factors, reduce ovarian tissue fibrosis and promote ovarian tissue angiogenesis; and promote the regeneration of senile ovarian follicles and ovarian endocrine function. Therefore, it has a good therapeutic and / or preventive effect on diseases related to mitochondrial dysfunction, especially in treating senile ovarian dysfunction.

[0032] Furthermore, the HEP14 drug of the present invention is a HEP14 / PLGA microsphere sustained-release formulation formed by encapsulating HEP14 with polylactic acid-glycolic acid copolymer. It has high HEP14 loading rate, encapsulation rate and long-lasting HEP14 sustained-release function. When using this microsphere sustained-release formulation for in vivo treatment, it not only solves the disadvantage of poor water solubility of HEP14, but also has few toxic side effects, so that HEP14 can exert a long-term and stable therapeutic effect in vivo, without the need for multiple administrations to the patient, thus improving the patient's medication compliance. Attached image description:

[0033] Figure 1 shows the transcriptomic profile of the ovaries of aged mice altered by HEP14.

[0034] Figure 2 shows how HEP14 restores mitochondrial autophagy and improves mitochondrial antioxidant stress function in senescent ovarian cells.

[0035] Figure 3 shows how HEP14 improves mitochondrial antioxidant stress function by enhancing mitochondrial autophagy in senescent ovarian cells.

[0036] Figure 4 shows how HEP14 enhances mitochondrial autophagy in senescent ovarian granulosa cell line KGN.

[0037] Figure 5 shows how HEP14 improves mitochondrial function in senescent KGN cells by enhancing mitophagy.

[0038] Figure 6 shows the function of HEP14 in enhancing mitophagy and mitochondrial ROS clearance by activating the PKC-ERK1 / 2 pathway.

[0039] Figure 7 shows how HEP14 improves the ovarian microenvironment in older women, thereby restoring ovarian follicle regeneration and development.

[0040] Figure 8 shows that HEP14 can significantly restore ovarian endocrine function in aged mice.

[0041] Figure 9 shows the morphological and property analysis of the HEP14 drug dosage form. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0044] Example 1: HEP14 alters the transcriptomic profile of the ovaries of aged mice

[0045] I. Collection of ovarian tissue samples

[0046] Four groups were included: (1) young mice aged 7–8 weeks; (2) adult mice aged 27–28 weeks; (3) aged mice aged 74–76 weeks treated with HEP14 solvent; and (4) aged mice aged 73–74 weeks treated with HEP14. HEP14 was diluted in 100 μL of physiological saline at a dose of 10 mg / kg body weight and administered via intraperitoneal injection every 5 days for a total of 5 injections. The control group received the same amount of physiological saline. After 28 days, ovarian tissue from one side was collected and cryopreserved at -80°C for later use.

[0047] II. RNA extraction, library construction and quality control, and transcriptome sequencing

[0048] First, after completing the standard RNA assay, RNA integrity and total amount were precisely detected using an Agilent 2100 bioanalyzer. Then, total RNA was used as the starting RNA for library construction. mRNA with polyA tails was enriched using Oligo(dT) magnetic beads, followed by random fragmentation of the mRNA with divalent cations in fragmentation buffer. Using the fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA was synthesized in an M-MuLV reverse transcriptase system. The RNA strand was then degraded with RNase H, and the second strand of cDNA was synthesized using dNTPs in a DNA polymerase I system. The purified double-stranded cDNA underwent end repair, A-tailing, and ligation with sequencing adapters. CDNA samples of approximately 370–420 bp were screened using AMPure XP beads, amplified by PCR, and the PCR products were purified again using AMPure XP beads to obtain the final library. After library construction, further library identification was performed. Preliminary quantification was conducted using a Qubit 2.0 Fluorometer, and the library was diluted to 1.5 ng / ul. Subsequently, the library was analyzed using an Agilent 2100 bioanalyzer. Once the insert size met expectations, the effective library concentration was accurately quantified using qRT-PCR (effective concentration higher than 2 nM) to ensure library quality. After passing the library analysis, sequencing was performed using an Illumina NovaSeq 6000, generating 150 bp paired-end reads.

[0049] III. Data Analysis

[0050] Principal component analysis (PCA) and column-clustered heatmaps showed that the transcriptomes of HEP14-treated aged (Aged_HEP14 mice) and adult mouse ovaries clustered together, but were significantly different from the transcriptomes of aged (Aged) and young (Young) mouse ovaries in the control group, and also significantly different from those in young (Young) mouse ovaries (Figures 1A and 1B). These results indicate that HEP14 significantly altered the transcriptomic profile of aged mouse ovaries, making it similar to that of adult mouse ovaries. Pearson correlation coefficient (PCC) analysis showed that the R² values ​​for each group were greater than 0.93 (Figure 1C), indicating minimal biological variability within the same group and providing a reliable basis for subsequent analysis. Volcano plots further revealed that, compared to aged control mouse ovaries, HEP14-treated senescent ovaries showed 4992 upregulated and 4901 downregulated differentially expressed genes (Figure 1D). Compared with the ovaries of adult mice in the control group, the ovaries of aged mice treated with HEP14 showed 1100 upregulated and 968 downregulated differentially expressed genes (Figure 1E). Gene ontology analysis revealed that, compared with the ovaries of aged mice in the control group, the differentially expressed genes in the ovaries of aged mice treated with HEP14 were enriched in biological functions such as autophagy (Figure 1F). Further gene set enrichment analysis (GSEA) results showed that, compared with the ovaries of aged mice in the control group, the pathways of "mitochondrial autophagy regulation," "oxidative stress response," "steroid hormone biosynthesis regulation," and protein kinase C (PKC) binding were significantly upregulated in the ovaries of aged mice treated with HEP14 (Figure 1G). In summary, the above data strongly support that HEP14 is involved in regulating signaling pathways such as mitophagy, "oxidative stress response," and "steroid hormone biosynthesis" in ovarian cells of aged mice, therefore, HEP14 treatment has the effect of improving ovarian function in aged mice.

[0051] Example 2: HEP14 restores mitochondrial autophagy and improves mitochondrial antioxidant stress function in senescent ovarian cells

[0052] Analysis of ovarian tissue transcriptome profiling (RNA-seq) data showed that, compared with the control group of adult mouse ovaries, HEP14 largely restored the expression of 54 genes related to mitophagy in HEP14-treated aging mouse ovarian cells (Figure 2A). The expression levels of autophagy markers Vdac1, autophagosome markers Map1lc3 and Sqstm1, and lysosome marker Lamp2 in HEP14-treated aged mouse ovarian tissue were randomly selected and examined. Consistent with the RNA-seq analysis, immunohistochemical staining and immunofluorescence double staining results showed that the expression levels of Map1lc3 and Sqstm1 proteins in aged mouse ovaries were significantly higher than those in the control group of aged mouse ovarian tissue (Figure 2B). Further triple-staining immunofluorescence staining and quantitative analysis of immunofluorescence images showed that in HEP14-treated aged ovarian tissue, the colocalization signal of Vdac1 with Map1lc3 and Lamp2 proteins was increased, significantly higher than that in the control group of aged mouse ovarian tissue (Figure 2C).

[0053] The results showed that HEP14 treatment increased mitophagy and the formation of mitophagosomes. In conclusion, HEP14 enhanced mitophagy in ovarian cells of aged mice.

[0054] Example 3: HEP14 improves mitochondrial antioxidant stress function by enhancing mitochondrial autophagy in senescent ovarian cells.

[0055] Age-related mitochondrial dysfunction in ovarian cells leads to excessive reactive oxygen species (ROS). RNA-seq analysis showed that HEP14 restored the mRNA expression levels of 49 genes related to mitochondrial function and oxidative stress in the ovaries of aged mice, reaching levels similar to those in adult mouse ovaries (Figure 3A). Immunohistochemical staining of ovarian sections and random detection of marker gene expression confirmed that, compared with control aged mouse ovaries, HEP14 treatment significantly increased the expression of antioxidant enzymes Catalase, Txnl1, and Hmox-1 in aged mouse ovarian tissue, while inhibiting the expression of the anti-apoptotic protein Bax (Figure 3B), suggesting that HEP14 treatment enhanced the antioxidant and anti-apoptotic capabilities of aged ovarian tissue. In conclusion, these results indicate that HEP14 improves the ovarian microenvironment by enhancing mitophagy and promoting mitochondrial antioxidant function, thereby promoting the regeneration and function of aged ovarian tissue.

[0056] Example 4: HEP14 enhances mitochondrial autophagy function in senescent ovarian granulosa cell line KGN cells

[0057] To restore ovarian physiological function, mitophagy promotes the clearance of dysfunctional mitochondria to prevent the accumulation of oxidative damage. This is a mitochondrial-dependent process. To elucidate the mechanism by which HEP14 promotes the recovery of ovarian function in senescent patients, this example first established a senescent granulosa cell model using the well-known ovarian granulosa cell line, KGN cells. Senescence of KGN cells was induced using the classic hydrogen peroxide (H2O2) stimulation method (referred to as senescent KGN cells). Compared with the control group treated with saline, KGN cells induced by 75 μM H2O2 showed high expression of senescence marker proteins SA-β-Gal and γ-H2AX, and reduced expression of the cell proliferation protein Ki67, confirming that 75 μM H2O2 induced KGN cell senescence (Figure 4A). The effect of HEP14 on the viability of senescent KGN cells was assessed using CCK8 reduction assays. The results showed that HEP14 treatment significantly restored the viability of senescent KGN cells compared to control cells (71.61±2.400% vs 58.69±1.280%). However, the mitophagy inhibitor midv-1 and the lysosomal inhibitor CQ (chloroquine) further reduced their viability to (55.16±1.155)% and (51.82±2.997)%, respectively (Figure 4B). These results suggest that HEP14-promoted mitophagy has a significant protective effect against H2O2-induced senescence of KGN cells. Furthermore, in senescent KGN cells, lysosomal LysoTracker Red and mitochondrial MitoTracker green staining showed increased LysoTracker red and MitoTracker green signals and co-localization signals (Figure 4C). More importantly, transmission electron microscopy (TEM) revealed a significant increase in the number of mitophagosomes, mitochondria, and lysosomes in HEP14-treated senescent KGN cells (Fig. 4D). These effects were inhibited by the mitophagy inhibitor Mdivi-1 or the lysosomal inhibitor CQ (chloroquine) (Fig. 4E). These results indicate that HEP14 promotes mitophagy function in senescent KGN cells. Furthermore, western blot results showed that HEP14 significantly increased the expression of autophagy marker proteins Map1lc3 and Vdac1 and decreased the expression of Sqstm1 in senescent KGN cells, indicating that mitophagy flux was inhibited, and that the mitophagy inhibitors and lysosomal inhibitors reversed these effects of HEP14 (Fig. 4F).

[0058] These results show that HEP14 promotes the formation of autophagosomes in senescent KNG cells and enhances mitochondrial autophagy in senescent KGN cells.

[0059] Example 5: HEP14 improves mitochondrial function in senescent KGN cells by enhancing mitophagy.

[0060] This study investigated whether enhanced mitophagy improves mitochondrial function by detecting mitochondrial membrane potential (ΔΨm) using JC1 (15,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzozolylcarbazine iodide) staining and analyzing antioxidant gene expression and reactive oxygen species levels using Western blotting. In HEP14-treated senescent KNG cells, the ratio of high-ΔΨm J-aggregates to low-ΔΨm JC-1 monomers was significantly higher than in the senescent model group (Figure 5A). Furthermore, in HEP14-treated senescent KNG cells, treatment with the mitophagy inhibitor Mdivi-1 and the lysosomal inhibitor chloroquine significantly decreased ΔΨm levels, indicating that HEP14 significantly increased ΔΨm levels in senescent KNG cells. This effect was significantly reversed by the mitophagy inhibitor Mdivi-1 and the lysosomal inhibitor chloroquine (Figure 5A). Western blot results showed that, compared with control senescent KGN cells, HEP14 upregulated the expression of key antioxidant genes Cat1 and Txnl1 in senescent KGN cells (Figure 5B). These regulatory effects of HEP14 were largely reversed by the mitophagy inhibitor Mdivi-1 and the lysosomal inhibitor chloroquine (Figure 5B). Furthermore, compared with untreated senescent KGN cells, ROS production in HEP14-treated senescent KGN cells was significantly reduced. Similarly, this effect was significantly reversed by Mdivi-1 and CQ (Figure 5C).

[0061] The results showed that HEP14 promoted ROS clearance by enhancing mitochondrial function. In conclusion, HEP14 enhances mitochondrial antioxidant stress response by activating mitophagy.

[0062] Example 6: HEP14 enhances mitophagy and mitochondrial ROS clearance by activating the PKC-ERK1 / 2 pathway.

[0063] RNA-seq analysis revealed differential gene enrichment in the PKC-binding signaling pathway in HEP14-treated aging mouse ovarian tissue (Figure 1G). MAPK1 / 3 (ERK1 / 2) expression was significantly increased in the ovaries of HEP14-treated aging mice compared to the control group (Figure 6A). Immunohistochemical staining of ovarian sections confirmed the RNA-seq results (Figure 6B). These results suggest that the PKC-ERK1 / 2 pathway may be essential for HEP14-activated mitophagy and improved mitochondrial function. PKC is an upstream regulator of the MAPK signaling pathway. Further investigation was conducted using an aging KGN cell model combined with the PKC inhibitor Go6850 and the ERK1 / 2 inhibitor U0126 to investigate the role of the PKC-ERK1 / 2 signaling pathway in promoting HEP14-enhanced mitophagy. Western blot analysis showed that HEP14 activated phosphorylation of PKC and ERK1 / 2, increased the expression levels of mitophagy marker proteins Map1lc3 and Vdac1, and decreased the protein level of Sqstm1. PKC and ERK1 / 2 inhibitors suppressed these effects of HEP14 (Figure 6C). Furthermore, MitoTracker green and LysoTracker red staining results showed that inhibition of ART and ERK1 / 2 significantly blocked HEP14-induced autophagosome formation in senescent KGN cells (Figure 6D). TEM analysis further confirmed these results (Figure E). ROS quantification results showed that HEP14 reduced ROS production in senescent KGN cells, and PKC and ERK1 / 2 inhibitors attenuated this effect of HEP14 (Figure 6F).

[0064] The results showed that HEP14 improved the function of aged ovarian cells by activating the PKC-ERK1 / 2 pathway, thereby restoring the function of mitophagy and mitochondrial ROS clearance in aged ovarian granulosa cells (GCs).

[0065] Example 7: HEP14 restores ovarian follicle regeneration and development by improving the ovarian microenvironment in older women.

[0066] I. HEP14 improves the microenvironment of nest tissue in aged mice

[0067] Ovarian development was assessed through histopathological examination (HE staining and IHC staining) and Picrosirius red (PSR) staining. Results showed that HEP14-treated mice exhibited significantly increased ovarian weight compared to control aged mouse ovaries (Figure 7A). PSR staining for tissue fibrosis revealed a significant decrease in fibrous collagen in the ovarian tissue of HEP14-treated aged mice compared to control aged mouse ovarian tissue (Figure 7B), decreased expression of α-smooth muscle actin (α-SMA), a typical marker of myofibroblasts (Figure 7C), and a significant increase in the expression of the vascular endothelial cell marker protein Vegfd (Figure 7D).

[0068] The results showed that HEP14 treatment reduced ovarian fibrosis in aging mice, promoted angiogenesis in ovarian tissue, and thus restored the microenvironment of ovarian tissue.

[0069] II. HEP14 promotes the regeneration and development of ovarian follicles in aged mice.

[0070] H&E staining and follicle counting analysis of ovarian sections showed that HEP14 treatment significantly increased the number of primordial follicles, preantral follicles, and antral follicles in aged ovaries compared to the control group (Fig. 7E). Similarly, in the ovaries of HEP14-treated aged mice, double immunofluorescence staining for Ki67 and Tunnel showed a significant increase in Ki67-positive and Tunnel-negative follicles, in stark contrast to the ovaries of the control group (Fig. 7F).

[0071] The results showed that HEP14 promoted the regeneration and development of follicles in older ovaries.

[0072] III. HEP14-activated PKC-ERK1 / 2-Stc1 pathway regulates follicular regeneration and development

[0073] RNA-seq analysis showed that key differentially expressed genes involved in follicle development and estrogen production were restored to levels close to those of adult ovaries (Fig. 7G). Immunohistochemical staining of ovarian sections not only confirmed HEP14-induced phosphorylation of PKC and increased expression and phosphorylation of ERK1 / 2 (Fig. 6B), but also confirmed increased expression levels of Stc1, ESR1, Amhr2, and Ptch1 proteins (Fig. 7H). Stc1 specifically induces granulosa cell growth in ovarian tissue, and granulosa cells recruit ovarian foam cells to growing follicles, thus promoting ovarian follicle regeneration and development. ESR1 and Amhr2 are granulosa cell markers. Ptch1 is an early marker protein for theca cell line differentiation. Western blot results confirmed that, compared with control senescent KGN cells, HEP14-treated senescent KGN cells showed significantly increased expression of Stc1, ESR1, and Amhr2 (Fig. 7I).

[0074] The results showed that HEP14 promoted follicle regeneration and development by activating the PKC-ERK1 / 2 pathway.

[0075] Example 8: HEP14 can significantly restore ovarian endocrine function in aged mice.

[0076] Ovarian endocrine function depends on follicular cells, with steroid production relying on steroid-producing cell-specific mitochondria to accommodate the specific enzymes required for steroid hormone biosynthesis. RNA-seq data are shown in Figure 1G. GSEA analysis revealed that differentially expressed genes were enriched in signaling pathways related to the regulation of steroid hormone biosynthesis in HEP14-treated aged mouse ovaries. After HEP14 treatment, the mRNA expression levels of 20 differentially expressed genes in this pathway recovered to levels similar to those in adult ovaries (Figure 8A). Immunohistochemical staining and Western blot analysis confirmed significantly increased levels of Vdac1 and Cyp1b1 proteins in HEP14-treated aged ovarian tissue and aged KGN cells (Figure B). More importantly, compared to the control group (Figure 8C), ELISA results showed significantly increased serum levels of ovarian gonadotropins E2, AMH, INHA, and INHB, but significantly decreased FSH levels, suggesting the recovery of ovarian endocrine function in HEP14-treated aged mice. Furthermore, it is noteworthy that the estrous cycle detection in mice showed that the estrous cycle of mice recovered after 4 weeks was close to that of adult mice in the control group, while the aged mice in the control group still showed irregular estrous cycles (Figure 8D).

[0077] The results showed that HEP14 treatment significantly improved the endocrine function of the ovaries in aged mice.

[0078] Example 9: Preparation and Identification of HEP14 Drug Dosage Form

[0079] In a dark environment, poly(lactic-co-glycolic acid) (PLGA) and HEP14 were dissolved in dichloromethane at a mass ratio of 5:1, and the mixture was stirred at 400 rpm using a magnetic stirrer. Next, the resulting solution was added to an aqueous solution containing 1% (w / v) polyvinyl alcohol (PVA), and the dichloromethane was allowed to evaporate at 25°C with magnetic stirring (300 rpm) for at least 12 hours. The microspheres were then collected by centrifugation (2000 × g, 5 min) and repeatedly washed with distilled water to thoroughly remove residual PVA. Finally, the microspheres were freeze-dried for 24 hours. This yielded a sustained-release formulation of HEP14 / PLGA microspheres encapsulated with PLGA.

[0080] Example 10: Preparation of HEP14 drug dosage form

[0081] In a dark environment, poly(lactic-co-glycolic acid) (PLGA) and HEP14 were dissolved in dichloromethane at a mass ratio of 10:1, and the mixture was stirred at 500 rpm using a magnetic stirrer. Next, the resulting solution was added to an aqueous solution containing 2% (w / v) polyvinyl alcohol (PVA), and the dichloromethane was allowed to evaporate at 25°C with magnetic stirring (500 rpm) for at least 12 hours. The microspheres were then collected by centrifugation (2000 × g, 5 min) and repeatedly washed with distilled water to thoroughly remove residual PVA. Finally, the microspheres were freeze-dried for 24 hours. This yielded a sustained-release formulation of HEP14 / PLGA microspheres encapsulated with PLGA.

[0082] Example 11: Identification of HEP14 / PLGA microspheres

[0083] For comparative analysis, we also prepared pure PLGA microspheres (HEP14-free PLGA microspheres) as an experimental control group using the same method as in Example 9.

[0084] To perform detailed morphological and property analysis of the microspheres prepared in Examples 9 and 10, as well as the experimental controls, we used scanning electron microscopy (SEM) to examine the samples. Simultaneously, we used ImageJ and Origin software for precise quantitative analysis of the microsphere particle size. Furthermore, we used an FTIR system to characterize the chemical structure of the samples. The scanning range was 400 to 4000 cm⁻¹. -1 Resolution is 4cm-1 The detection was performed at a scanning speed of 64 scans / min, and a total of 1867 data points were obtained.

[0085] To further investigate the crystal structure characteristics of the samples, we analyzed the microspheres using X-ray diffraction (XRD). The XRD patterns of the samples were collected using a Rigaku Smartlab X-ray diffractometer. During the experiment, we used a Cu Kα radiation source (45 kV, 120 mA) and set the 2θ angle range to 5°–100°. The obtained data were collected as transmittance values ​​(%) and processed and analyzed using Oringin software.

[0086] SEM analysis revealed that both PLGA and HEP14 / PLGA microspheres exhibited uniform size distribution, spherical morphology, and smooth surfaces (Figures 9A and 9B). Due to the high loading of HEP14, the particle size of the 5:1 HEP14 / PLGA microspheres (prepared in Example 9) (0.1688 μm ± 0.0685) and the 10:1 HEP14 / PLGA microspheres (prepared in Example 10) (0.100 μm ± 0.0735) were significantly increased compared to pure PLGA microspheres (0.052 μm ± 0.028). To verify whether the HEP14 drug was successfully encapsulated within the PLGA, infrared spectroscopy analysis was performed on drug-loaded and unloaded microspheres. The results showed that no vibrational peak of HEP14 was observed in the infrared spectrum of the HEP14 / PLGA sample (Figure 9C). This suggests that HEP14 may be completely encapsulated within the PLGA matrix, or that the vibrational peaks of the PLGA matrix are similar to those of HEP14, thus masking the HEP14 signal. X-ray diffraction analysis revealed multiple diffraction peaks in HEP14, indicating its inherent crystalline properties (Figure 9D). In the HEP14 / PLGA mixture, the diffraction peaks of HEP14 disappeared, indicating a decrease in the crystallinity of HEP14 during loading. This change allows HEP14 to be gradually released from the PLGA microspheres, making it easier for organisms to absorb and utilize.

[0087] To determine the drug loading rate, encapsulation efficiency, and in vitro release of HEP14 / PLGA microspheres, we used a UV spectrophotometer to measure the drug loading and encapsulation efficiency of the HEP14 / PLGA microspheres and plotted a standard curve of OD value (Y) and concentration (c) of the HEP14 / PLGA microspheres. The detection wavelength was 201 nm, and the sample volume was 1 mL. The drug loading (%) and encapsulation efficiency (%) were calculated using the following formulas, where the theoretical drug loading of the microspheres refers to the initial amount of drug used to prepare the microspheres. Drug loading rate (%) = (weight of drug in microspheres) / (weight of microspheres) × 100%; Retention rate (%) = (actual drug loading in microspheres) / (theoretical drug loading in microspheres) × 100%. The in vitro release rate of HEP14 / PLGA / microspheres was measured at 201 nm using a UV spectrophotometer. HEP14 / PLGA microspheres were placed in PBS solution at pH 7.4 and incubated at 37°C and 100 rpm in a shaker. The microspheres were precipitated by centrifugation (5000×g, 3 min), and the supernatant was collected at 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 days for analysis.

[0088] To investigate the drug loading, encapsulation efficiency, and release rate of HEP14, a standard curve was obtained at the maximum absorption peak at 201 nm: y = 0.0785c + 0.1555 (R² = 0.9966, n = 6). The linear correlation coefficient R² reached 0.99853, fully meeting the requirements for determining the drug loading and content of microspheres. The HEP14 / PLGA microspheres prepared in Example 9 had a drug loading and encapsulation efficiency of 3.10% and 18.04%, respectively. The HEP14 / PLGA microspheres prepared in Example 10 had a drug loading and encapsulation efficiency of 2.1% and 26.02%, respectively. The in vitro release kinetics of HEP14 / PLGA microspheres were determined by ultraviolet spectrophotometry (Figure 9E). Initially, rapid release occurred on the first day, followed by sustained and controlled release from day 1 to day 9. Subsequently, additional bursts of release were observed from day 9 to day 17, eventually leading to gradual release until equilibrium was reached on day 17. At the end of the study, the cumulative release of HEP14 / PLGA microspheres was approximately 9.73%.

[0089] In summary, the HEP14 drug formulations prepared in Examples 9 and 10, namely the HEP14 / PLGA microsphere sustained-release formulations, have the functions of high HEP14 drug loading rate, encapsulation rate and long-acting sustained release of HEP14.

[0090] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0091] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Application of HEP14 in the preparation of drugs that activate protein kinase C (PKC).

2. Application of HEP14 in the preparation of drugs that induce and activate extracellular regulatory protein kinase 1 / 2 (ERK1 / 2).

3. Application of HEP14 in the preparation of drugs that promote mitochondrial generation.

4. Application of HEP14 in the preparation of drugs that promote mitophagy and mitochondrial function.

5. Application of HEP14 in the preparation of drugs that promote the secretion of antioxidant enzymes and scavenge reactive oxygen species.

6. Application of HEP14 in the preparation of drugs for treating tissue fibrosis.

7. Application of HEP14 in the preparation of drugs that promote tissue angiogenesis.

8. The application according to any one of claims 1 to 7, characterized in that, The tissue in question is ovarian tissue.

9. Application of HEP14 in the preparation of drugs that promote the growth of ovarian follicular granulosa cells.

10. Application of HEP14 in the preparation of drugs that promote hormone secretion from ovarian follicle granulosa cells.

11. Use of HEP14 in the preparation of drugs for the treatment and / or prevention of diseases related to mitochondrial dysfunction.

12. Application of HEP14 in the preparation of drugs for treating ovarian dysfunction in the elderly.

13. Application of HEP14 in the preparation of drugs that promote the regeneration of ovarian follicular cells.

14. Application of HEP14 in the preparation of drugs for restoring ovarian endocrine function.

15. A dosage form of HEP14, characterized in that, The formulation is a HEP14 / PLGA microsphere sustained-release formulation formed by encapsulating HEP14 with polylactic acid-glycolic acid copolymer (PLGA).

16. The HEP14 dosage form according to claim 15, characterized in that, The HEP14 / PLGA microspheres have a smooth surface.

17. The HEP14 dosage form according to claim 16, characterized in that, The HEP14 / PLGA microspheres have a particle size of 0.0265–0.2373 micrometers.

18. The HEP14 dosage form according to claim 17, characterized in that, The HEP14 / PLGA microspheres have a particle size of 0.1003–0.2373 micrometers.

19. The HEP14 dosage form according to claim 15, characterized in that, The HEP14 loading rate in the HEP14 / PLGA microspheres is 2.1% to 3.1%.

20. The HEP14 dosage form according to claim 15, characterized in that, The encapsulation efficiency of HEP14 in the HEP14 / PLGA microspheres is 18.04% to 26.02%.

21. A method for preparing an HEP14 drug dosage form, characterized in that, Includes the following steps: In a dark environment, polylactic acid-glycolic acid copolymer (PLGA) and HEP14 were dissolved in dichloromethane at a mass ratio of 5–10:1, and stirred with a magnetic stirrer at a speed of 400–500 rpm. Then, the resulting solution was added to an aqueous solution containing 1–2% (w / v) polyvinyl alcohol, and stirred with a magnetic stirrer at a speed of 300–500 rpm at 25°C to allow the dichloromethane to evaporate for at least 12 hours. Then, the microspheres were collected by centrifugation and repeatedly washed with distilled water to completely remove residual polyvinyl alcohol. Finally, the microspheres were placed in a freeze dryer for freeze-drying to obtain the HEP14 / PLGA microsphere sustained-release formulation formed by polylactic acid-glycolic acid copolymer encapsulating HEP14.

22. The method according to claim 21, characterized in that, The polylactic acid-hydroxyacetic acid copolymer and HEP14 were dissolved in dichloromethane at a mass ratio of 5:1.

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