Application of deoxycholic acid and analogues thereof
By pretreating cells with deoxycholic acid and its analogs, Parthanatos cell death was significantly inhibited, and the problem of PARP-1 inhibitors in the prior art affecting cell self-repair function was solved, and a safe and effective Parthanatos inhibition effect was achieved.
Patent Information
- Application Number
- CN202510021608.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-05-09
AI Technical Summary
When existing PARP-1 inhibitors inhibit Parthanatos cell death, they affect the self-repair function of cells, resulting in serious side effects and are difficult to protect damaged neurons for a long time.
Using deoxycholic acid and its analogues, Parthanatos cell death was significantly inhibited by pretreatment of cells, with a final concentration of 10-200 μM.
Deoxycholic acid and its analogs can effectively inhibit Parthanatos cell death, and its inhibitory effect is different from the mechanism of action of existing PARP-1 inhibitors, and does not affect the DNA repair function of the cells and avoid side effects.
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Abstract
Description
Technical Field
[0001] The present invention relates to application of deoxycholic acid and its analogs, and in particular to application of deoxycholic acid and its analogs in inhibiting Parthanatos cell death. Background Art
[0002] When PARP-1 in cells is in an overactivated state, it will cause the cells to die in a new way. In this process, PAR polymers are both cell death signals and mediators, so this mode of death is also called Parthanatos. Parthanatos is a new form of programmed cell death.
[0003] The death mechanism of Parthanatos includes DNA damage, overactivation of PARP-1, NAD + and ATP depletion, nuclear translocation of AIF and MIF complexes, and DNA fragmentation. For example, ionizing radiation, exposure to n-methyl-d-aspartate (NMDA) or DNA alkylating agent n-methyl-n0-nitro-n-nitrosoguanidine (MNNG), oxygen glucose deprivation (OGD), or long-term treatment with hydrogen peroxide and other reactive oxygen species (ROS) can directly induce DNA damage, leading to PARP-1 overactivation and the production of long-chain and branched PAR polymers. The formation of PAR consumes NAD in cells. + As a reaction substrate, NAD+ resynthesis requires about 2-4 ATP molecules to further consume endogenous functional molecules. PAR produced by PARP-1 overactivation is released from the nucleus into the cytoplasm, where it binds to apoptosis-inducing factor (AIF), causing AIF to be released from the mitochondria. Once AIF is released from the outer surface of the mitochondria into the cytoplasm, it binds to macrophage migration inhibitory factor (MIF) and transfers to the nucleus together. MIF is a DNA nuclease that cleaves genomic DNA into large fragments (approximately 20-50kb fragments), resulting in chromatin and nuclear condensation fragments, which in turn triggers cell death.
[0004] PARP1 is the first protein discovered to have PAR (poly ADP ribose) modification activity. It can synthesize linear and branched PAR polymers using NAD+ as a substrate, and modify some nuclear proteins and itself. PARP1 has dual functions: one is the protective effect. PARP1 can recruit some DNA repair-related proteins through PAR modification to promote cell survival by playing a key role in DNA base excision repair, single-strand break repair and alternative non-homologous end joining to prevent the accumulation of potentially lethal DNA double-strand breaks. The second is the aforementioned role in promoting cell death, that is, PARP1 promotes DNA breakage by promoting the translocation of AIF from mitochondria to the nucleus and activating the nuclease activity of MIF, which then triggers cell death, thereby eliminating defective cells that are considered unsalvageable to maintain the health of the entire body.
[0005] The upstream event of Parthanatos is the overactivation of PARP-1. Although PARP-1 inhibitors can significantly inhibit Parthanatos to prevent cell death and have become drugs, they are still mainly used to inhibit the DNA repair function of PARP-1 to treat cancer. It is precisely because of the dual function of PARP-1 that while blocking Parthanatos by inhibiting PARP-1 to protect cells, it also affects the self-repair function of cells. Therefore, PARP-1 inhibitors have serious side effects in animal models of Parthanatos-related diseases.
[0006] Previous studies have attempted to use PARP1 inhibitors to protect damaged neurons. PARP1 inhibitors do have the effect of maintaining the survival of nerve cells during use. However, since the activity of PARP1 in recruiting DNA repair proteins is also inhibited, damaged DNA cannot be properly repaired. Therefore, once the PARP1 inhibitor is removed, cells that have accumulated a large amount of DNA damage will continue to undergo Parthanatos. This also limits the use of PARP1 inhibitors in clinical applications as neuroprotective drugs. Therefore, there is an urgent need to develop new drugs that protect functional cells by inhibiting the function of PARP1 downstream proteins. Blockers targeting Parthanatos are of great application value in the treatment of related diseases.
[0007] Deoxycholic acid is also known as deoxycholic acid (Deoxycholic acid, 3α, 12α-Dihydroxy-5β-cholanicacid, 7-Deoxycholic acid) CAS number: 83-44-3. It is a white or off-white crystalline powder, odorless. Melting point (℃): 176-178℃. Specific rotation (°): [α] D20 + 55° (C = 1, in ethanol). Solubility: Solubility at 15℃ (g / L): 0.24 in water, 200.7 in ethanol, 1.16 in ether, 2.94 in chloroform, 10.46 in acetone, 9.06 in glacial acetic acid. Soluble in alkali hydroxide and carbonate solution. Deoxycholic acid is a bile acid lacking a hydroxyl group on C-7. It is a free bile acid derived from bile acid losing an oxygen atom. It mainly exists in the form of taurine and glycine in bile.
[0008] Deoxycholic acid has the activity of promoting bile secretion, promoting food digestion and absorption, and eliminating bile stasis. It has been successfully used in the treatment of biliary diseases and lipomas. Studies have also shown that deoxycholic acid can act as a tumor stimulating factor, triggering inflammation, intestinal metaplasia, cancer, promoting cell apoptosis, etc., and is involved in the occurrence and development of many diseases such as gastric diseases, intestinal diseases, and esophageal diseases. The mechanism of deoxycholic acid promoting the occurrence of colorectal cancer mainly includes: (1) Inducing calcium signal transduction and then regulating the mitogen-activated protein kinase-epidermal growth factor receptor (EGFR-MAPK) signaling pathway. Under the stimulation of deoxycholic acid, the intracellular calcium ion level is significantly increased, the calcium signaling pathway is activated, and then the expression of calmodulin-related kinase II (CAMKII) and the activation of tyrosine kinase (c-Src) are induced, leading to the activation of mitogen-activated protein kinase (MAPK) signaling, activating the proto-oncogene activated protein-1 (AP-1) and inhibiting the tumor suppressor gene P53, promoting the occurrence of colorectal cancer. (2) Acting on the amalgamator-metalloproteinase 17 / epidermal growth factor receptor (ADAM-17 / EGFR) signaling axis to regulate intestinal carcinogenesis. (3) Activating the Wnt / β-catenin pathway and upregulating the expression of β-catenin protein, accelerating the mitosis and metastasis of colorectal cancer cells. (4) Activating the cyclooxygenase-2 / prostaglandin 2 (COX-2 / PEG2) signaling pathway, mediating the proliferation and invasion of colorectal cancer epithelial cells. (5) It can cause abnormal mitosis of colon cells and transform them into cancer cells, and can also destroy the stability of the genome, including oxidative damage to DNA, chromosomes, mitochondria and endoplasmic reticulum. (6) Acting on mitochondria to promote the death of colorectal epithelial cells. Studies have shown that deoxycholic acid induces oxidative stress in human colon cell mitochondria and causes cell damage. (7) Inducing the occurrence of colorectal cancer by downregulating the expression of miR-199a-5p or increasing the expression of CAC1 protein. (8) Activation of vascular endothelial growth factor receptor 2 (VEGFR2) promotes the formation of vascular mimicry and epithelial-mesenchymal transition (EMT), thereby exacerbating the occurrence of colorectal cancer. (9) It can promote the proliferation of colon cancer cells, and long-term (7 days) deoxycholic acid stimulation can significantly enhance the migration and invasion of colon cancer cells. Its mechanism may be related to the upregulation of N-cadherin expression and the downregulation of vitamin D receptor (VDR) expression.
[0009] Deoxycholic acid is associated with the occurrence and development of bile duct diseases. Its main mechanisms are as follows: (1) It increases the activity of scar fibroblasts, acts on fibroblasts, produces a large amount of collagen, and finally thickens the duct wall. It also converts fibroblasts into myofibroblasts, promoting the occurrence of bile duct spasm. (2) Deoxycholic acid increases the secretion of cholesterol and affects the nucleation process of cholesterol stones. In addition, deoxycholic acid can also promote the secretion of various protein components such as mucin and IgA in bile, which promotes the occurrence of cholelithiasis. (3) It may reduce the expression of epithelial cadherin (E-cadherin) in hepatobiliary carcinoma cells (RBE) by binding to G protein-coupled receptors (TGR-5), while increasing the expression of transcription factors Snail, N-cadherin, and vimentin. It may also promote the metastasis of bile duct carcinoma cells by inducing EMT in RBE cells, thereby promoting the invasion of bile duct carcinoma cells. Previous studies have also found that deoxycholic acid is involved in the pathogenesis of other diseases. For example, deoxycholic acid can inhibit the proliferation of neuroblastoma cells by promoting the dephosphorylation of protein kinase B, upregulating Bax protein expression, reducing Bcl-2 protein expression, increasing ROS content, and activating Caspase-3; subcutaneous injection of deoxycholic acid can cause hemolysis, which may be related to its damage to red blood cell permeability; increased deoxycholic acid levels may lead to vascular calcification, aggravating cardiovascular and kidney diseases, etc. There are no reports of an association between deoxycholic acid and Parthanatos. Summary of the invention
[0010] Purpose of the invention: The purpose of the present invention is to provide the use of deoxycholic acid and its analogs in inhibiting the death of Parthanatos cells, so as to solve the problem of how to inhibit the death of Parthanatos cells. Another purpose of the present invention is to propose the use of deoxycholic acid and its analogs in the preparation of drugs for inhibiting the death of Parthanatos cells, so as to solve the problem of how to prepare drugs for inhibiting the death of Parthanatos cells.
[0011] Technical solution: Use of the deoxycholic acid or a pharmaceutically acceptable salt thereof in the present invention in inhibiting the death of Parthanatos cells.
[0012] Preferably, the final concentration of the deoxycholic acid or a pharmaceutically acceptable salt thereof is 10-200 μM. Pretreatment of cells with deoxycholic acid can significantly inhibit the death of Parthanatos cells.
[0013] Another aspect of the present invention discloses the use of deoxycholic acid analogs or pharmaceutically acceptable salts thereof in inhibiting the death of Parthanatos cells.
[0014] Preferably, the deoxycholic acid analogue comprises at least one of taurodeoxycholic acid, glycodeoxycholic acid, sodium deoxycholate, ursodeoxycholic acid, and chenodeoxycholic acid.
[0015] Preferably, the final concentration of the deoxycholic acid analog or a pharmaceutically acceptable salt thereof is 10-200 μM.
[0016] The third aspect of the present invention discloses the use of deoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting Parthanatos cell death.
[0017] The fourth aspect of the present invention discloses the use of the above-mentioned deoxycholic acid analogs or pharmaceutically acceptable salts thereof in the preparation of drugs for inhibiting Parthanatos cell death.
[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:
[0019] The present invention discloses for the first time that deoxycholic acid and its analogs can inhibit Parthanatos cell death, and among these substances, deoxycholic acid has the strongest inhibitory effect. The present invention also confirms that the blocking effect of deoxycholic acid on Parthanatos is not achieved by inhibiting the activity of PARP-1, which is completely different from the mechanism of action of existing blockers targeting Parthanatos, and can effectively circumvent the defects of existing blockers, providing an important reference for the subsequent development of drugs for inhibiting Parthanatos cell death. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 Cell microscopic pictures of different concentrations of deoxycholic acid inhibiting Parthanatos cell death;
[0021] Figure 2 The effect of different concentrations of deoxycholic acid on cell viability;
[0022] Figure 3 The inhibitory effects of different inhibitors on Parthanatos cell death;
[0023] Figure 4 and Figure 5 Cell microscopic pictures of different concentrations of deoxycholic acid analogs inhibiting Parthanatos cell death;
[0024] Figure 6 The effect of deoxycholic acid on PARP1 activity. DETAILED DESCRIPTION
[0025] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.
[0026] Unless otherwise specified, the culture medium used in each embodiment is DMEM culture medium containing 10% by mass concentration of fetal bovine serum.
[0027] The solutions of each substance in each example were obtained by diluting each substance to a specified concentration using a DMEM culture medium containing 10% by mass concentration of fetal bovine serum.
[0028] The cultures involved in each embodiment were all cultured in a cell culture incubator at 37° C., 5% CO 2 in air and 100% humidity.
[0029] Example 1: Deoxycholic acid was used to inhibit the death of Parthanatos cells, the method is as follows:
[0030] MNNG was selected to induce the classic cell model of Parthanatos in Hela cells, and the final concentration of MNNG used in the model was 50 μM.
[0031] In order to verify that the cell death model constructed by MNNG-induced Hela cells was PARthanatos, the following experiment was conducted: Hela cells were pretreated with DPQ (PARP1 inhibitor) at a final concentration of 30 μM, ZVAD (apoptosis inhibitor) at a final concentration of 10 μM, 3MA (autophagy death inhibitor) at a final concentration of 10 mM, and N-1 (necrosis inhibitor) at a final concentration of 10 μM for 24 hours, and then MNNG was added at a final concentration of 50 μM for further treatment for 15 minutes. The cells were washed three times with culture medium, and then 100 μL of culture medium was added for further culture for 24 hours. The cell viability was detected using the CCK8 kit. The results are shown in Figure 2. Figure 3 As shown, the blank control group was not given medication or modeling treatment, while the MNNG group was only given modeling treatment. Figure 3 It was shown that only PARP1 inhibitors could inhibit cell death, while apoptosis inhibitors, necrosis inhibitors and autophagic death inhibitors could not inhibit cell death, which proved that the PARthanatos model was successfully constructed.
[0032] Based on the PARthanatos model, the protective effect of deoxycholic acid was verified. The structural formula of deoxycholic acid is:
[0033]
[0034] Hela cells were pretreated with deoxycholic acid solution of gradient concentration (final concentration was 0, 12.5, 25, 50, 100, 200 μM) for 24 hours, and the cell morphology was observed under a microscope. The deoxycholic acid solution was discarded, and then 50 μM MNNG solution (prepared by dissolving MNNG in cell culture medium) was added for further 15 minutes. The cells were washed with culture medium for 3 times, and then 100 μL culture medium was added for further 24 hours. The cell morphology was observed under a microscope. The results are as follows: Figure 1 As shown, Figure 1In the figure, DMSO represents the control group with only dimethyl sulfoxide as the solvent, DCA represents deoxycholic acid treatment, and MNNG represents the modeling using MNNG.
[0035] After observing the cell morphology, CCK8 was added to detect cell viability. The results are as follows Figure 2 As shown, Figure 2 Blank is the blank control group (control group with only solvent dimethyl sulfoxide), MNNG is the model group, DPQ is the PARP1 inhibitor positive control group, and 12.5μM, 25μM, 50μM, 100μM, and 200μM represent different final concentrations of deoxycholic acid treatment groups. Figure 1 and Figure 2 It can be seen that deoxycholic acid can inhibit the occurrence of Parthanatos in a dose-dependent manner and reduce cell death. Pretreatment with deoxycholic acid at a concentration of more than 50 μM for 24 hours has a very obvious protective effect on MNNG-induced Parthanatos cell death.
[0036] Example 2: Application of deoxycholic acid analogs to inhibit MNNG-induced Parthanatos, the method is as follows:
[0037] After confirming that deoxycholic acid has a significant inhibitory effect on Parthanatos in Hela cells, we further tested whether the four structural analogs of deoxycholic acid also have the effect of blocking Parthanatos, namely Ursodeoxycholic acid (UDCA), Chenodeoxycholic Acid (CDCA), Isodeoxycholic acid (IDCA), and Deoxycholic acid sodium salt (DCAS). The structural formulas are as follows:
[0038]
[0039]
[0040] After determining the appropriate working concentration of each deoxycholic acid analog on Hela cells through preliminary experiments, Hela cells were pretreated with gradient concentrations (final concentrations of 0, 12.5, 25, 50, 100, and 200 μM) of UDCA / CDCA / IDCA / DCAS solutions for 24 hours, and then added with MNNG solution for modeling (final concentration of 50 μM, 15 minutes). The cells were washed three times with culture medium, and then 100 μL of culture medium was added to each well for a further 24 hours. The cell morphology was observed under a microscope. The results are as follows: Figure 4 and Figure 5As shown, the results showed that under the condition of MNNG toxicity, UDCA and CDCA could also inhibit the death of Parthanatos caused by MNNG treatment to a certain extent, but the effect was not as significant as deoxycholic acid.
[0041] Example 3: Deoxycholic acid does not prevent the occurrence of Parthanatos by inhibiting PARP-1
[0042] In order to determine whether the blocking effect of deoxycholic acid on Parthanatos is achieved by inhibiting PARP-1, the present invention detected the expression level of PAR, a product of PARP-1 (using a Western blotting method, using a PAR-modified antibody to detect the expression level of PAR, a product of PARP-1).
[0043] Hela cells were pretreated with 50 μM PARP-1 inhibitor olapamil (OLA) and gradient concentrations (final concentrations of 25, 50, 100, and 200 μM) of deoxycholic acid solution for 24 hours, and then 50 μM MNNG solution (prepared by dissolving MNNG in cell culture medium) was added for another 30 minutes. Cells in each group were collected for WB experiments. Figure 6 As shown, Figure 6 WT represents the DMSO-only control group, DCA25, DCA50, DCA100, and DCA200 represent 25, 50, 100, and 200 μM deoxycholic acid treatments, respectively, MNNG represents the Parthanatos model group, and OLA represents the positive drug group.
[0044] Figure 6 It shows that the production of PAR induced by MNNG was inhibited after pretreatment with OLA, and OLA significantly inhibited the activity of PARP1. However, pretreatment with deoxycholic acid at different concentrations, which inhibits the action of Parthanatos in cells, did not affect the production of PAR, which indicates that the blocking effect of deoxycholic acid on Parthanatos is not through the inhibition of PARP-1 activity. In other words, deoxycholic acid inhibits PARthanatos but does not affect the activity of PARP1, and thus does not affect the DNA damage repair function.
[0045] The present invention uses the classic cell model of Parthanatos induced by MNNG in Hela cells to determine that deoxycholic acid, sodium deoxycholate, ursodeoxycholic acid, and chenodeoxycholic acid can inhibit the death of Parthanatos cells, and deoxycholic acid has the strongest inhibitory effect.
Claims
1. Use of deoxycholic acid or a pharmaceutically acceptable salt thereof in inhibiting Parthanatos cell death.
2. The use according to claim 1, characterized in that: The final concentration of the deoxycholic acid or its pharmaceutically acceptable salt is 10-200 μM.
3. Use of a deoxycholic acid analog or a pharmaceutically acceptable salt thereof in inhibiting Parthanatos cell death.
4. The use according to claim 3, characterized in that: The deoxycholic acid analogs include at least one of taurodeoxycholic acid, glycodeoxycholic acid, sodium deoxycholate, ursodeoxycholic acid, and chenodeoxycholic acid.
5. The use according to claim 3, characterized in that: The final concentration of the deoxycholic acid analog or a pharmaceutically acceptable salt thereof is 10-200 μM.
6. Use of deoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting Parthanatos cell death.
7. Use of a deoxycholic acid analog or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting Parthanatos cell death.