A method for protecting glaucoma RGCs by regulating USP14 expression

Abstract

The present invention provides a method for protecting glaucoma RGCs by regulating USP14 expression, and belongs to the technical field of glaucoma prevention. The method for protecting glaucoma RGCs by regulating USP14 expression of the present invention comprises dissolving the USP14 small molecule inhibitor IU1 in a solvent to prepare an IU1 buffer; applying the IU1 buffer to RGCs to protect the RGCs; wherein: the USP14 small molecule inhibitor IU1 protects RGCs by promoting the expression of anti-apoptosis-related Bal‑2 protein in RGCs; the USP14 small molecule inhibitor IU1 protects RGCs by reducing the expression of pro-apoptosis-related Bax protein in RGCs. The method of the present invention can accurately and effectively protect RGCs, thereby achieving the technical effect of preventing or treating glaucoma.

Description

Technical Field

[0001] The present invention relates to the technical field of glaucoma prevention, and in particular to a method for protecting glaucoma RGCs by regulating USP14 expression. Background Art

[0002] Glaucoma is the world's leading irreversible blinding eye disease. Progressive death of retinal ganglion cells (RGCs) is the main pathological basis of glaucomatous optic neuropathy. It is currently believed that high intraocular pressure is the main risk factor for glaucoma and the only clinically treatable cause. However, even if confirmed patients are given intraocular pressure-lowering treatment, it cannot prevent the secondary death of RGCs and damage to their axons. The visual function of some affected eyes continues to deteriorate. It is estimated that within 20 years, 27% of patients will eventually become blind in at least one eye and 10% of patients will eventually become blind in both eyes [1]. The pathogenesis of glaucoma is unclear and there is currently no effective intervention method. Therefore, further exploring the death mechanism of glaucoma RGCs and finding new RGCs protection measures remain the direction of efforts in this field.

[0003] More and more studies have found that mitochondrial autophagy dysfunction may be closely related to the damage of RGCs in glaucoma, and enhancing mitochondrial autophagy through various pathways can reduce RGCs damage. Parkin is an E3 ubiquitin ligase that mediates the ubiquitination of mitochondrial outer membrane proteins and participates in mitochondrial autophagy. When mitochondria are damaged, their membrane potential decreases, causing the cytoplasmic parkin protein to aggregate on the mitochondrial outer membrane. Parkin will ubiquitinate mitochondrial outer membrane proteins, further recruit the autophagy receptor optineurin protein to bind to mitochondria, and induce the downstream autophagy-related protein LC3 to wrap mitochondria to form autophagosomes for degradation through lysosomes. Evidence has shown that enhancing parkin-mediated mitochondrial autophagy promotes the survival of neurons in Parkinson's disease and Alzheimer's disease, and plays an important role in neuroprotection in neurodegenerative diseases.

[0004] However, mitochondrial autophagy has two sides, and uncontrolled mitochondrial autophagy may lead to massive cell death. The applicant's previous study found that in the early stage of the chronic ocular hypertension rat model (on the third day of ocular hypertension), mitochondrial autophagy was overactivated, and the up-regulated expression of mitochondrial autophagy-related parkin may play a protective role in RGCs by weakening parkin-mediated mitochondrial autophagy; and as the intraocular pressure increased, on the 14th day of ocular hypertension, mitochondrial autophagy function was impaired. The up-regulated expression of parkin or mitochondrial fusion-related OPA1 may promote the parkin-mediated mitochondrial autophagy pathway, alleviate the mitochondrial autophagy dysfunction caused by high ocular pressure, and then play a protective role in RGCs. This suggests that mitochondrial autophagy within the relatively normal range plays an important role in maintaining the normal function of RGCs. However, our research on parkin-mediated mitochondrial autophagy in RGCs of experimental glaucoma models is still in its infancy, and the specific mechanism of signal molecule interaction needs further exploration. Why the up-regulated expression of parkin plays a dual regulatory role in mitochondrial autophagy in the damage of RGCs mitochondria at different stages remains to be further explored.

[0005] The delicate dynamic balance of ubiquitination and deubiquitination is used to regulate the homeostasis of mitophagy in vivo. Ubiquitinating / deubiquitinating enzymes (DUBs) antagonize parkin by opposing ubiquitination. Ubiquitin-specific protease (USP) is the largest family of DUBs and changes in many neurodegenerative diseases. Therefore, USP has gradually become a potential key pharmacological target for regulating the ubiquitin mechanism for the protection of neurons. Among the three DUBs related to mammalian proteasomes (USP14, UCH37 and RPN11), USP14 is a very attractive target because its substrate deubiquitination is very fast and USP14 has a unique ability to regulate proteasome complex activity and autophagy. Therefore, some scholars believe that compared with inhibiting other DUBs, inhibiting USP14 may have greater advantages in promoting mitophagy. They found that in the fruit fly model of PINK1 / Parkin mutation, downregulating USP14 expression can promote mitophagy and thus improve mitochondrial dysfunction. In addition, the commercialization of IU1, a specific small molecule inhibitor of USP14, makes USP14 an ideal candidate target for the treatment of diseases. IU1 has been shown to protect neurons by regulating the mitochondrial autophagy pathway in models of neurodegenerative diseases such as Parkinson's disease and stroke. As can be seen from the above, USP14 may be developed as a potential therapeutic target for neurodegenerative diseases by regulating mitochondrial autophagy. Optic neuropathy in glaucoma is also a type of degenerative disease, which suggests that USP14 may play a neuroprotective role on RGCs in glaucoma. However, whether USP14 can regulate parkin-mediated mitochondrial autophagy and reduce the damage of RGCs in glaucoma models has not been reported yet.

[0006] So, how does USP regulate mitophagy? Studies have found that USP8 and USP33 regulate mitophagy by directly deubiquitinating parkin. The difference is that downregulating USP8 inhibits mitophagy by delaying the translocation of parkin to depolarized mitochondria, while downregulating USP33 enhances mitophagy by promoting the translocation of parkin to depolarized mitochondria. However, USP15, USP30, and USP35 do not antagonize the ubiquitination state of parkin itself, but antagonize the ubiquitination of mitochondrial outer membrane proteins mediated by parkin. Further exploration revealed that the mitochondrial outer membrane translocase TOM20 (translocase of outer mitochondrial membrane, also known as TOMM20), as an important subunit of the mitochondrial outer membrane translocase, was found to be one of the substrates of USP30. At the same time, TOM20 is also a substrate of parkin and a specific ubiquitination site of parkin. In addition, studies have shown that USP30 can offset parkin-mediated mitophagy activity by regulating the ubiquitination level of TOM20. The above suggests that the effect of enhancing mitophagy by only upregulating the expression of parkin may not be strong and lasting enough, because USP in the cell can also downregulate parkin-mediated mitophagy through various pathways. It can be seen that the use of USP14 to regulate parkin-mediated mitophagy may provide a new treatment option for neuroprotection in glaucoma. However, it is unclear what specific mechanism USP14 uses to affect mitophagy, and it is impossible to achieve precise regulation of USP14 to achieve preventive protection against glaucoma.

[0007] Therefore, providing a method to protect glaucomatous RGCs by regulating USP14 expression has important practical significance. Summary of the invention

[0008] The purpose of the present invention is to provide a method for protecting glaucoma RGCs by regulating USP14 expression, aiming to solve the technical problem that the influence mechanism of USP14 on mitochondria is unclear in the prior art, and it is impossible to accurately regulate USP14 and thus protect glaucoma RGCs.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a method for protecting glaucoma RGCs by regulating USP14 expression, comprising:

[0011] The USP14 small molecule inhibitor IU1 is dissolved in a solvent to prepare an IU1 buffer solution;

[0012] Applying the IU1 buffer to RGCs to protect the RGCs;

[0013] in:

[0014] The USP14 small molecule inhibitor IU1 protects RGCs by promoting the expression of anti-apoptosis-related Bal-2 protein in RGCs;

[0015] The USP14 small molecule inhibitor IU1 protects RGCs by reducing the expression of pro-apoptosis-related Bax protein in RGCs.

[0016] Furthermore, the solvent is DMSO.

[0017] Furthermore, the concentration of the IU1 buffer is 100 μM.

[0018] The present invention also provides the use of the method for protecting glaucoma RGCs by regulating USP14 expression as described in the above technical solution in the prevention of glaucoma.

[0019] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0020] The method of the present invention uses the USP14 small molecule inhibitor IU1 to prepare a solution of a certain concentration to regulate the relevant gene proteins in RGCs and thus achieve protection of RGCs. The method is accurate and efficient and can prevent existing glaucoma with higher efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the analysis diagram of the retinal immunoblotting results of the blank group, control group and experimental group in test example 1;

[0022] Figure 2 This is the result diagram of fluorescent gold retrograde labeling of the blank group, control group and experimental group in test example 1;

[0023] Figure 3 This is a schematic diagram of the effect of IU1 on the viability of RGCs in RGCs culture medium in Test Example 2;

[0024] Figure 4 This is a Western blot analysis of IU1 on RGCs in a control environment and a glutamate excitotoxicity model in Test Example 2;

[0025] Figure 5 This is a schematic diagram of the effect of IU1 on RGCs in the control environment and glutamate excitotoxicity model in Test Example 2;

[0026] Figure 6Schematic diagram of confocal microscopy observation after mitochondria in RGCs in Test Example 2 are labeled red with MitoTracker dye;

[0027] Figure 7 This is a diagram showing the effect of down-regulated Parkin expression in IU1 on RGCs in the glutamate excitotoxicity model in Test Example 2 and the results of immunoblotting analysis. DETAILED DESCRIPTION

[0028] The present invention provides a method for protecting glaucoma RGCs by regulating USP14 expression, comprising:

[0029] The USP14 small molecule inhibitor IU1 is dissolved in a solvent to prepare an IU1 buffer solution;

[0030] Applying the IU1 buffer to RGCs to protect the RGCs;

[0031] in:

[0032] The USP14 small molecule inhibitor IU1 protects RGCs by promoting the expression of anti-apoptosis-related Bal-2 protein in RGCs;

[0033] The USP14 small molecule inhibitor IU1 protects RGCs by reducing the expression of pro-apoptosis-related Bax protein in RGCs.

[0034] In the present invention, the solvent is preferably DMSO.

[0035] In the present invention, the concentration of the IU1 buffer is preferably 100 μM.

[0036] The present invention also provides the use of the method for protecting glaucoma RGCs by regulating USP14 expression as described in the above technical solution in the prevention of glaucoma.

[0037] In the present invention, unless otherwise specified, the required raw materials for preparation are all commercially available products well known to those skilled in the art.

[0038] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0039] Test Case

[0040] Verify the effect of USP14 small molecule inhibitor IU1 on RGCs

[0041] In vitro experiments on RGCs and preliminary experiments on rats with ocular hypertension were conducted. Specifically, IU1 4.7μM, 25μM, 100μM and 500μM was used in the in vitro experiments on RGCs, and the mitochondrial membrane potential was measured using JC-1 to confirm that the optimal drug concentration for the cell model was 100μM. Then, IU1 was used for 30min, 6h and 24h in the control experiment, and the mitochondrial membrane potential was measured using JC-1 to confirm that the optimal drug time for the cell model was 30 minutes. The preliminary experiment on rats with ocular hypertension was as follows: IU1 4.7μM and 100μM were injected into the vitreous cavity, and 100μM was determined by a preliminary experiment using fluorescent gold retrograde labeling.

[0042] The results are shown in Table 1. Based on Table 1, it can be seen that the intravitreal injection concentration of IU1 is 100 μM. The intraocular pressure of all laser modeled rats increased (P < 0.01), and there was no significant statistical difference in the mean and peak intraocular pressure between rats injected with IU1 and rats injected with control drugs.

[0043] Table 1. Record of the effects of RGCs in vitro experiments and pre-experiments on intraocular pressure in rats

[0044]

[0045] The blank group, control group and experimental group were analyzed by retinal immunoblotting. Figure 1 As shown, based on Figure 1 It can be seen that compared with the blank group (rat with high intraocular pressure without modeling), the expression level of retinal GFAP protein in the control drug group injected with intravitreal injection increased at 14 days of high intraocular pressure (P < 0.01, and the expression level of retinal Bax protein also increased (P < 0.01), while the expression level of Bcl-2 protein decreased (P < 0.01). Compared with the control group, the expression level of retinal GFAP protein in the IU1 group injected with intravitreal injection decreased at 14 days of high intraocular pressure (P < 0.01), and the expression level of retinal Bax protein also decreased (P < 0.01), while the expression level of Bcl-2 protein increased (P < 0.05). Therefore, intravitreal injection of IU1 reduces the expression of GFAP protein induced by retinal Müller cells, that is, reduces the activation of Müller cells. In addition, IU1 reduces the expression of pro-apoptosis-related Bax protein and increases the expression of anti-apoptosis-related Bcl-2 protein.

[0046] The blank group, control group and experimental group were retrogradely labeled with fluorescent gold. The results are as follows: Figure 2 As shown, based on Figure 2It can be seen that compared with the blank group (rats without modeling of high intraocular pressure), the number of surviving retinal RGCs in the central, mid-peripheral and peripheral parts of rats with high intraocular pressure for 14 days in the vitreous cavity injection control drug group was significantly reduced (n=6, P<0.01). Compared with the control group, the number of surviving retinal RGCs in the central, mid-peripheral and peripheral parts of rats with high intraocular pressure in the vitreous cavity injection IU1 group was significantly increased.

[0047] In summary, IU1 plays a protective role in RGCs in the chronic ocular hypertension rat model.

[0048] Test Example 2

[0049] Different concentrations of IU1 were prepared using 60 mg / mL DMSO as the solvent as the experimental group, and 60 mg / mL DMSO was used as the control group to verify that IU1 had a positive effect on the survival ability of RGCs in the RGCs culture medium. The results are as follows Figure 3 shown; based on Figure 3 It can be seen that IU1 has a positive effect on the viability of RGCs in the RGCs culture medium, and the optimal concentration of IU1 on RGCs is 100 μM, and the optimal duration is 30 minutes;

[0050] Western blot analysis of IU1 on RGCs in control and glutamate excitotoxicity models showed the following results: Figure 4 Knowable; based on Figure 4 It can be seen that in the RGCs culture medium, the percentage of apoptotic cell death measured by Hoechst staining was found to be reduced in IU1-treated RGCs compared with the control group (P < 0.05). Cytotoxicity measured by LDH activity showed that RGCs treated with IU1 exhibited lower cytotoxicity (P < 0.01). Mitochondrial membrane potential was measured by JC-1 and it was found that RGCs treated with IU1 exhibited higher mitochondrial membrane potential (P < 0.01). The expression levels of USP14 and Bax proteins were decreased (P < 0.05), while the expression level of Parkin protein and the ratio of LC3-II to LC3-I were increased (P < 0.05). However, no statistical differences were observed in the expression levels of Bcl-2 and LAMP1 proteins in IU1-treated RGCs compared with the control group;

[0051] In summary, IU1 can enhance the survival ability of RGCs in RGCs culture medium.

[0052] Verify the effect of IU1 on RGCs in the control environment and glutamate excitotoxicity model. Figure 5 As shown, based on Figure 5It can be seen that the proportion of apoptotic cells in IU1-treated RGCs was significantly reduced compared with that in the control group treated with 100 μM glutamate (P < 0.05). These results indicate that IU1 can reduce apoptosis in the glutamate excitotoxic model. Cytotoxicity measured by LDH activity also showed a similar trend. In the glutamate excitotoxic environment, the amount of LDH released by RGCs increased compared with the normal RGCs culture medium, however, the effect of IU1 treatment reversed the LDH release (P < 0.05). These results indicate that IU1 can reduce cytotoxicity in the glutamate excitotoxic model. In the glutamate excitotoxic environment, the JC-1 ratio of the control group was 0.87 ± 0.03, while the JC-1 ratio of RGCs after IU1 treatment was 0.95 ± 0.03, which was significantly higher than before (P < 0.05). These results indicate that IU1 can increase mitochondrial membrane potential under glutamate excitotoxicity. Under glutamate excitotoxicity, the expression level of Bax protein in RGCs treated with IU1 was decreased (P < 0.01), and the expression level of Bcl-2 protein was increased (P < 0.05) compared with the control group. These results indicate that IU1 can increase the expression of anti-apoptotic Bcl-2 protein and reduce the expression of pro-apoptotic Bax protein under glutamate excitotoxicity;

[0053] The expression level of USP14 protein in IU1-treated RGCs was decreased compared with that in the control group, both in RGCs culture medium and under glutamate excitotoxic environment, indicating that IU1 can effectively inhibit USP14 expression in the in vitro glutamate excitotoxic model (P < 0.05). In addition, under glutamate excitotoxic environment, the ratio of LC3-II to LC3-I (P < 0.05) and the expression levels of optineurin and LAMP1 (P < 0.05) proteins in IU1-treated RGCs were increased compared with those in the control group. The expression levels of parkin (P < 0.05) and TOM20 (P < 0.05I) proteins in IU1-treated RGCs were increased compared with those in the control group, both in RGCs culture medium and under glutamate excitotoxic environment.

[0054] In summary, IU1 can protect RGCs in the glutamate excitotoxicity model.

[0055] The mitochondria in RGCs were labeled with MitoTracker dye in red and observed under a confocal microscope. Figure 6 shown; based on Figure 6It can be seen that in the control group under the glutamate excitotoxicity model, small spherical mitochondria were distributed in RGCs around the nucleus, while few mitochondria were observed in axons. The mean fluorescence intensity of MitoTracker was significantly increased in IU1-treated RGCs, and a larger number of large mitochondria were observed around the nucleus, partially restoring the tubular mitochondrial network.

[0056] In summary, IU1 can effectively inhibit USP14 expression and increase Parkin-mediated mitophagy-related proteins and TOM20 expression in the glutamate excitotoxicity model, suggesting that the Parkin-mediated mitophagy pathway is also upregulated.

[0057] Effects of down-regulated Parkin expression in IU1 on RGCs in the glutamate excitotoxicity model and the results of immunoblotting analysis Figure 7 shown; based on Figure 7 It can be seen that by immunoblotting, whether in RGCs culture medium or in the environment of 100μM glutamate, the LC3-II to LC3-I ratio (P<0.05), optineurin (P<0.05) and LAMP1 (P<0.05) protein expression levels in RGCs treated with Parkin siRNA+IU1 were reduced compared with those in the siRNA control group+IU1 treatment group. In the RGCs culture medium, the TOM20 protein expression level in RGCs treated with Parkin siRNA+IU1 was significantly lower than that in the siRNA control group+IU1 treatment group (P<0.05), but no significant statistical difference was found in the environment of 100μM glutamate.

[0058] The results further confirmed that the neuroprotective effect of IU1 on RGCs in the glutamate excitotoxicity model was achieved by enhancing the parkin-mediated mitochondrial autophagy pathway.

[0059] In summary, the present invention explores the principle involved in "the specific mechanism by which USP14 affects mitochondrial autophagy is unclear, and it is impossible to accurately regulate USP14 to achieve preventive protection against glaucoma" recorded in the background technology, that is, the neuroprotective effect of IU1 on RGCs in the glutamate excitotoxicity model is achieved by enhancing the parkin-mediated mitochondrial autophagy pathway. Furthermore, the method of protecting glaucomatous RGCs by regulating USP14 expression described in the present invention can effectively protect RGCs, thereby preventing or treating glaucoma.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for protecting glaucoma RGCs by regulating USP14 expression, characterized in that: include: The USP14 small molecule inhibitor IU1 is dissolved in a solvent to prepare an IU1 buffer solution; Applying the IU1 buffer to RGCs to protect the RGCs; in: The USP14 small molecule inhibitor IU1 protects RGCs by promoting the expression of anti-apoptosis-related Bal-2 protein in RGCs; The USP14 small molecule inhibitor IU1 protects RGCs by reducing the expression of pro-apoptosis-related Bax protein in RGCs.

2. The method for protecting glaucoma RGCs by regulating USP14 expression according to claim 1, characterized in that: The solvent is DMSO.

3. The method for protecting glaucoma RGCs by regulating USP14 expression according to claim 1, characterized in that: The concentration of the IU1 buffer was 100 μM.

4. Use of the method for protecting glaucoma RGCs by regulating USP14 expression as described in any one of claims 1 to 3 in the prevention or treatment of glaucoma.

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