USP10 inhibits the apoptosis of lens epithelial cells and delays the progression of diabetic cataract via the deubiquitination and stabilization of MCL1

Abstract

As one of the complications of diabetes mellitus (DM), diabetic cataract (DC) has become the critical cause of vision impairment. MCL1 is an antiapoptotic protein in the BCL2 family that plays an important role in cell survival and proliferation. The role of MCL1 in DC remains unclear. Exploring the function of MCL1 and its underlying regulatory mechanism in DC can provide new prevention ideas. Here, high-glucose (HG)-cultured lens epithelial cells (LECs) and streptozotocin (STZ)-induced diabetic model rats were used. MTT assays, western blotting, immunohistochemical (IHC) assays and propidium iodide (PI) staining were utilized to analyse LECs apoptosis. Immunofluorescence staining was used to assess ROS levels in LECs in a high-glucose environment. Overexpression experiments and co-IP analyses were performed to assess the protein‒protein interaction between MCL1 and USP10. The results showed that HG induced apoptosis and oxidative stress in LECs and induced cataract in diabetic rats. MCL1 overexpression inhibited HG-induced apoptosis. Moreover, USP10 stabilized the MCL1 protein by binding to and deubiquitinating MCL1, and this binding was attenuated in HG environments. Furthermore, the antioxidant (-)-Epigallocatechin-3-gallate (EGCG) significantly delayed cataract progression in diabetic rats by reversing oxidative stress-induced LECs apoptosis in a HG environment. In summary, our experiments revealed that USP10 inhibited LECs apoptosis and the occurrence of DC in a HG environment by deubiquitination and stabilization of MCL1. The antioxidant EGCG significantly delayed cataract progression in diabetic rats by reversing oxidative stress-induced LECs apoptosis in a HG environment. Our study helps elucidate the molecular mechanism of DC and provides new therapeutic targets and ideas for the subsequent development of nonsurgical treatment options.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-47681-6.

Introduction

With the incidence of diabetes mellitus (DM) increasing annually worldwide, diabetic eye complications, such as diabetic cataract¹(DC) and diabetic retinopathy, have become the leading cause of visual impairment in diabetic patients^2,3. Currently, surgery is the mainstay of treatment for DC⁴. Owing to the postoperative complications of surgery⁵, exploring new therapeutic treatments for DC is still an important task.

The pathogenesis of DC is still not fully clear. Multiple mechanisms may contribute to the development of DC, such as increased oxidative stress⁶, apoptosis and autophagy of lens epithelial cells (LECs)⁷, activation of the polyol pathway⁸, and hypertonic and epithelial-mesenchymal transition⁹. In particular, oxidative stress is known to play a significant role in the development of DC since it can lead to damage to lens proteins and lipids. The regulation of LECs apoptosis is also thought to be important in the pathogenesis of cataract¹⁰.

MCL1, an antiapoptotic protein in the BCL2 family, exerts its antiapoptotic effects in many types of cells¹¹. Compared with other proteins in the BCL2 family, MCL1 has a shorter half-life and instability due to its PEST domains, leading to the rapid degradation of the MCL1 protein and therefore promoting cell apoptosis¹¹. The view that MCL1 expression is downregulated in age-related cataract patients has been confirmed^1,12. However, the expression and related regulatory mechanism of MCL1 in DC are still unclear.

Previous studies have shown that the stability of the MCL1 protein is tightly controlled by protein modification processes such as ubiquitination, phosphorylation, and acetylation^13,14. Ubiquitination is a widespread protein posttranslational modification that plays a crucial role in regulating various cellular functions¹⁵. In the ubiquitin-proteasome pathway, E3 ligases can directly induce the polyubiquitination and degradation of the MCL1 protein. Conversely, deubiquitinases such as USP9X¹⁶, USP13¹⁷, and USP20¹⁸ have been reported as interacting partners of MCL1, which may stabilize MCL1 by removing ubiquitin from the MCL1 protein, thereby inhibiting apoptosis.

Ubiquitination is a widespread protein post-translational modification that plays a crucial role in regulating various cellular functions¹⁵. However, the ubiquitination process is reversible and can be done by a series of deubiquitinases. Ubiquitin-specific peptidase 10 (USP10) is a member of the ubiquitin-specific protease family that removes the ubiquitin chain from ubiquitin-conjugated protein substrates and thus regulate multiple important biological processes such as tumor cell growth, cellular immunity, and apoptosis¹⁹. Since MCL1 is an antiapoptotic protein in the BCL2 family, whether USP10 is involved in regulating the stability of the MCL1 protein requires further research.

(-)-Epigallocatechin-3-gallate (EGCG) is the most commonly found catechin in green tea, accounting for 60% of the total catechin content²⁰. Owing to its structural properties, EGCG has high antioxidant and radical scavenging capacities²¹. Multiple studies have shown that EGCG can treat various systemic diseases through its antioxidant properties^22,23, especially in diabetic patients. Whether EGCG can play a role in the treatment of DC through its antioxidant effect evoke our interest.

In this study, we found that USP10 inhibited LECs apoptosis and the occurrence of DC in the HG environment by deubiquitination and stabilization of MCL1. The antioxidant EGCG significantly delayed cataract progression in diabetic rats by reversing oxidative stress-induced LECs apoptosis in the HG environment. Our study contributes to elucidating the molecular mechanism of DC and provides new therapeutic targets and ideas for the subsequent development of nonsurgical treatment options.

Materials and methods

Cell lines and cell culture

The human LEC lines HLE-B3 and SRA01/04 were obtained from Guang Zhou Jennio Biotech Co., Ltd. (Guangzhou, China). HLE-B3 cells were cultured in minimum essential medium containing 10% foetal bovine serum, and SRA01/04 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5.5 mM glucose and 5% foetal bovine serum (both from Gibco; Thermo Fisher Scientific Inc., Waltham, MA, USA). The cells were grown in monolayer cultures at 37 °C in a humidified atmosphere consisting of 5% CO2.

Cell viability assay

Cells were seeded in 96-well plates at a density of 2.0 × 10³ cells/well and were then treated with the indicated concentrations of glucose on the second day. The cells were cultured with glucose for 24, 48 or 72 h and then subjected to MTT assays. Each group of cells was incubated with 20 µL of MTT (5 mg/mL) (Sigma–Aldrich; Merck KGaA) for 3 h at 37 °C. Then, the solution was discarded, and 200 µL of dimethyl sulfoxide was added. The absorbance of formazan at 495 nm was measured using an ELISA Multiskan reader (Thermo Fisher Scientific, Inc.). The data were recorded and analysed, and the results are presented as means ± SDs.

Western blot analysis

Whole-cell protein lysates were prepared and analysed by western blotting in accordance with a previously described protocol²⁴. After being harvested and rinsed with prechilled PBS, the cells were lysed, and the extract was centrifuged at 13,200 × g at 4 °C for 15 min. The protein concentrations were measured using the BSA assay (Beyotime, Shanghai, China). Whole-cell protein lysates (40 µg) were electrophoresed on 12% denaturing polyacrylamide slab gels and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore. Ltd.) through electroblotting. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then probed with specific primary antibodies against USP10 (D7A5, 1:1000), MCL1 (sc-12756, 1:1000), Caspase9 (9502s, 1:500), Caspase3 (NB100-56708, 1:1000), PARP (1:500) and GAPDH (TA-08, 1:2000) at 4 °C overnight, followed by probing with secondary antibodies for 2 h at room temperature. Antibody binding was detected using an enhanced chemiluminescence (ECL) system (EMD Millipore, Billerica, MA, USA) in accordance with the manufacturer’s protocol. The protein expression levels were quantified using ImageJ software (version 1.6.0_24; National Institute of Health, Bethesda, MD, USA).

Cell apoptosis assay

PI staining was used to assess the level of apoptosis in cells. A propidium iodide (PI) apoptosis detection kit was purchased from BIO-BOX Biotech (Nanjing, China). The cells were treated with HG (50 mM) for 72 h, after which the culture medium was discarded, and the cells were subsequently washed with prechilled PBS. Five hundred microlitres of binding buffer was added to each well. The cells in each group were subsequently incubated with 5 µL of PI for 15 min in the dark at room temperature. Nuclei were visualized using Hoechst. Images were obtained using a laser scanning confocal microscope (Leica Microsystems GmbH).

Plasmid transfection

The pcDNA3.1-Mcl-1-C-His plasmid, pENTER-USP10-Flag plasmid and pcDNA3.1-UBB-C-Flag plasmid were obtained from Biosune Biotechnology (Shanghai, China). HLE-B3 and SRA01/04 cells were seeded in 6-well plates and transfected with plasmids using jetPRIME^® in vitro DNA & siRNA transfection reagent (Polyplus, France) in accordance with the manufacturer’s protocol. Then, the cells were treated with the indicated concentration of glucose for 48 h and subjected to subsequent analyses.

Coimmunoprecipitation assays

HLE-B3 and SRA01/04 cells were transfected with plasmids (pcDNA3.1-Mcl-1-C-His, pENTER-USP10-Flag or pcDNA3.1-UBB-C-Flag) and treated with the indicated concentration of glucose for 48 h. Then, the cells were washed 2 times in PBS and lysed with cell lysis buffer for IP. The protein concentrations were measured using the BSA assay (Beyotime, Shanghai, China). For the His-tag or Flag-tag immunoprecipitation assay, 1000 µg of whole-cell lysates were incubated with 6 µL of His-tag monoclonal antibody (66005-1-Ig, Proteintech Group) or 10 µL of Flag-tag mouse monoclonal antibody (AF2852, Beyotime, Shanghai, China) at room temperature for 0.5 h. Then, 20 µL of beads was added, and the mixture was incubated at room temperature for 2 h in a rotating incubator. The beads were washed 3 times with 1× lysis/wash buffer (Absin, Shanghai, China). After being diluted with SDS loading buffer, the beads were boiled for subsequent western blotting. Normal mouse immunoglobin G (IgG) or rabbit IgG (Millipore) was used as the negative control.

Immunofluorescence staining

Intracellular ROS levels in cells were measured using a Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China) with 2’,7’‑dichlorodihydrofluorescein diacetate (DCFH‑DA) in accordance with methods described below. After high-glucose treatment, the culture medium was discarded, and the cells were subsequently washed with PBS twice and incubated with DCFH‑DA (0.5 µM) in the dark for 30 min at 37 °C. Nuclei were visualized using Hoechst. Finally, the cells were washed again with PBS. Images were obtained using a laser scanning confocal microscope (Leica Microsystems GmbH).

Establishment of the diabetic rat models

All animal experiments were performed following the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research under the ethics approval of the Animal Ethics Committee of Shandong Provincial Hospital. Furthermore, the study was conducted in accordance with the ARRIVE guidelines. Six-week-old male Wistar rats were purchased from Shandong Pengyue Laboratory Animal Technology Co. ,Ltd. and raised in a specific pathogen-free environment and provided adequate water and food. The rats were adaptively fed for 2 weeks. The diabetic rat model was established by the intraperitoneal injection of STZ (65 mg/kg in citrate buffer), and the standard for the confirmation of diabetes was defined as a blood glucose level higher than 16.7 mmol/L at 72 h after STZ injection. Diabetic rats were randomly divided into a DM group and an EGCG group. The rats in the EGCG group were treated by daily oral administration of aqueous solution dissolved with EGCG at a dose of 200 mg/kg body weight, and the anterior segments of all eyes were photographed using a slit lamp every three weeks after the models were successfully generated. Rats were anesthetized for 20 min with 30 mg/kg sodium pentobarbital via intraperitoneal injection (IPI) when taking the photos. Finally, the animals were euthanized with an overdose of sodium pentobarbital, and the eyeballs were removed from the rats for immunohistochemical analysis at week 9.

Immunohistochemical analysis

After cataract formation was evaluated at 9 weeks, the rats were sacrificed. Eye balls were harvested, fixed in FAS Eye Tissue Fixative Solution (Servicebio Technology Company, Wuhan, China), and then embedded in paraffin. Then, the lens tissues were cut to a thickness of 4 μm (Leica HistoCore MULTICUT, Germany) and used for immunostaining. The primary antibodies used in the immunohistochemistry assays were as follows: anti-USP10 (D7A5, 1:100) and anti-MCL1 (66026-1-Ig, 1:400).

Statistical analysis

All the experiments were repeated at least three times. All the statistical analyses were performed using SPSS statistical software (version 20.0; IBM Corp., Armonk, NY, USA). The data are presented as means ± SDs of at least three independent assays performed in duplicate or triplicate. An unpaired t test was used to compare two groups, and one-way ANOVA was used to compare more than two groups. P values less than 0.05 were considered significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001).

Results

HG induced LECs apoptosis

To investigate the cytotoxicity of HG to LECs, SRA01/04 and HLE-B3 cells were cultured in medium supplemented with six different glucose concentrations for 24 h, 48 h, and 72 h. The effect of HG on cell viability was analysed using MTT assays. The results demonstrated that epithelial cell viability significantly decreased with increasing concentrations of glucose (Fig. S1A). These results suggested that HG inhibited the survival of LECs in a concentration-dependent manner. Considering the chronic pathogenicity of DC and the degree of survival inhibition of LECs in the HG environment in this study, three concentrations of glucose, 25 mM, 50 mM, 100 mM, and 72 h were selected as the experimental variables for subsequent experiments.

To further explore the effect of HG on LECs apoptosis, the expression of apoptosis-related proteins was verified by western blot assays. After 72 h of exposure to HG medium containing 0 to 100 mM glucose, the protein levels of cleaved-caspase9, cleaved-caspase3 and PARP increased in a concentration-dependent manner (Fig. 1A, B). Furthermore, the effect of the HG environment on LECs apoptosis was verified by PI staining (Fig. 1C). In summary, HG decreased the survival and promoted the apoptosis of LECs in a dose-dependent manner.

Fig. 1.

Expression levels of apoptosis-related proteins in LECs in a HG environment. (A) HLE-B3 and SRA01/04 cells were treated with 0, 25, 50, or 100 mM glucose for 72 h. The expression of caspase9, caspase3 and PARP was assessed by western blot assays. GAPDH was used as a reference protein. (B) Relative level of cleaved-PARP adjusted to the level of GAPDH (ns: not statistically significant, *P < 0.05, **P < 0.01). (C) Lens epithelial cells were treated with 0, 25, 50, or 100 mM glucose for 72 h. PI staining was used to assess the degree of cell apoptosis. Hoechst was used to stain nuclei.

MCL1 inhibited HG-induced LECs apoptosis

As an antiapoptotic protein in the BCL2 family, MCL1 plays a key role in cell survival. To evaluate the effect of MCL1 on LECs apoptosis induced by HG, we first examined the expression of MCL1 in LECs in the high-glucose environment. Compared with the control conditions, the HG environment inhibited the expression of the MCL1 protein in a concentration-dependent manner (Fig. 2A, B). We subsequently overexpressed MCL1 by transfecting the pcDNA3.1-Mcl-1-C-His plasmid into SRA01/04 and HLE-B3 cells. The overexpression of MCL1 significantly attenuated the increased expression of cleaved caspase3 and PARP induced by HG (Fig. 2C, D). To validate the above results, PI staining was performed. The results showed that incubation in the HG environment increased cell death, an effect that was rescued by the overexpression of MCL1 (Fig. 2E). These results suggest that MCL1 plays an important role in the HG-induced apoptosis of LECs.

Fig. 2.

MCL1 inhibits high glucose-induced apoptosis. (A) HLE-B3 and SRA01/04 cells were treated with 0, 25, 50, or 100 mM glucose for 72 h. The expression of MCL1 was assessed by western blotting. GAPDH was used as a reference protein. (B) Relative level of MCL1 adjusted to the level of GAPDH (ns: not statistically significant, *P < 0.05, **P < 0.01). (C) HLE-B3 and SRA01/04 cells were treated with glucose (50 mM) and transfected with a pcDNA3.1-Mcl-1-C-His plasmid for 72 h. The expression of MCL1, caspase3 and PARP was assessed by western blot assays. GAPDH was used as a reference protein. (D) Relative level of cleaved-PARP adjusted to the level of GAPDH (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). (E) HLE-B3 and SRA01/04 cells were transfected with a pcDNA3.1-Mcl-1-C-His plasmid and then treated with glucose (50 mM) for 72 h. PI staining was used to assess the degree of cell apoptosis. Hoechst was used to stain nuclei.

MCL1 expression was downregulated by HG via the ubiquitin‒proteasome pathway

Since MCL1 has a shorter half-life and instability due to its PEST domains, the stability of the MCL1 protein is tightly controlled by protein modification processes such as ubiquitination. To verify whether MCL1 expression is affected by ubiquitination levels in HG-treated cells, we inhibited the ubiquitin‒proteasome pathway with MG132 and found that MCL1 protein levels were significantly increased, suggesting that the downregulation of MCL1 protein expression induced by HG was due to the proteasome pathway (Fig. 3A, B).

Fig. 3.

HG induces the ubiquitination and degradation of the MCL protein in LECs. (A) HLE-B3 and SRA01/04 cells were treated with glucose (50 mM) or MG132 (2 µL) for 72 h. The expression of MCL1 was assessed by western blot assays. GAPDH was used as a reference protein. (B) Relative level of MCL1 adjusted to the level of GAPDH (*P < 0.05). (C) His-MCL1 and Flag-UBB were transfected into HLE-B3 and SRA01/04 cells, and immunoprecipitation was then performed with an anti-His antibody. Ubiquitinated MCL1 was analysed using an in vitro ubiquitination assay.

To test our hypothesis, UBB plasmids were transfected into HLE-B3 and SRA01/04 cells, and immunoprecipitation experiments were performed to measure the level of MCL1 ubiquitination. It was showed that HG treatment increased the level of ubiquitinated MCL1 in Fig. 3C. These above results confirmed that the HG environment increased the ubiquitination level of the MCL1 protein, thereby inducing LECs apoptosis.

USP10 interacts with and stabilizes MCL1 by deubiquitination

USP10 is a deubiquitination enzyme that has been reported to regulate the stability and activity of BCL-2 family proteins. To determine whether USP10 is involved in HG-induced MCL1 proteasomal degradation, we first assessed the expression of USP10 in SRA01/04 and HLE-B3 cells cultured in glucose-containing medium. The results revealed that the expression of the USP10 protein decreased in the SRA01/04 and HLE-B3 cell lines while the glucose concentration in the medium increased (Fig. 4A, B). We then overexpressed USP10 in LECs and found that overexpressing USP10 significantly increased MCL1 protein levels (Fig. 4C). Correspondingly, cleaved-caspase3 and PARP protein levels were reduced. These findings suggest that USP10 could inhibit the apoptosis of LECs in the HG environment by regulating MCL1.

Fig. 4.

USP10 participates in LECs apoptosis in a HG environment. (A) HLE-B3 and SRA01/04 cells were treated with 0, 25, 50, or 100 mM glucose for 72 h. The expression of USP10 and GAPDH was assessed by western blot assay. (B) Relative level of USP10 adjusted to the level of GAPDH (*P < 0.05). (C) LECs were transfected with the pENTER-USP10-Flag plasmid and then treated with glucose (50 mM) for 72 h. The expression of USP10, MCL1, Caspase3, PARP and GAPDH was assessed by western blot assays. (D) LECs were exposed to CHX (2µM) at 0, 2, 4, 6, and 8 h after treatment with glucose (50 mM) and transfected with the pENTER-USP10-Flag plasmid. The expression of USP10, MCL1 and GAPDH was assessed by western blot assays. (E) Degradation curve for MCL1 in the SRA01/04 and HLE/B3 cell lines (**P < 0.01, ***P < 0.001).

CHX, a protein synthesis inhibitor, was subsequently added to two HG-treated cell groups, one of which overexpressed USP10. LECs were collected at separate time points (0, 2, 4, 6, and 8 h) for western blotting. The results showed that the overexpression of USP10 delayed the degradation of the MCL protein in the HG environment (Fig. 4D, E), which verified that USP10 may be involved in the posttranslational regulation of MCL1.

To confirm whether USP10 can deubiquitinate and therefore stabilize MCL1, protein coimmunoprecipitation analysis was performed after cotransfecting Flag-tagged USP10 and His-tagged MCL1 into LECs. MCL1 was detected in USP10 immunoprecipitates using anti-Flag antibodies (Fig. 5A). USP10 was also detected in MCL1 immunoprecipitates using anti-His antibodies in a similar manner (Fig. 5B). In addition, the HG environment inhibited the binding of USP10 to the MCL1 protein (Fig. 5A, B).

Fig. 5.

USP10 interacts with and deubiquitinates MCL1. (A) His-MCL1 and Flag-USP10 proteins were co-overexpressed in LECs, followed by immunoprecipitation with an anti-His antibody. Coimmunoprecipitated USP10 was assessed using an anti-USP10 antibody. (B) His-MCL1 and Flag-USP10 proteins were co-overexpressed in LECs, followed by immunoprecipitation with an anti-Flag antibody. Coimmunoprecipitated MCL1 was detected using an anti-MCL1 antibody.

Taken together, our preliminary results revealed that MCL1 is a potential USP10 substrate in LECs. The HG environment weakened the binding of USP10 to MCL1, thereby reducing the level of MCL1 deubiquitination and ultimately inducing the degradation of MCL1 and the apoptosis of LECs.

The antioxidant EGCG reversed oxidative stress-induced LEC apoptosis in the HG environment

Oxidative stress is known to play a significant role in the development of DC⁶. We assessed the effect of HG on cellular oxidative stress. LECs were cultured in medium supplemented with different concentrations of glucose for 72 h, and immunofluorescence staining revealed that the ROS levels increased with increasing glucose concentration (Fig. 6A).

Fig. 6.

EGCG inhibits oxidative stress induced by HG. (A) ROS levels were assessed by DCFH-DA in SRA01/04 and HLE-B3 cells after culture in medium with different glucose concentrations for 72 h. (B) EGCG (10 µM), glucose (50 mM) or EGCG (10 µM)+glucose (50 mM) was added to the medium of SRA01/04 and HLE-B3 cells, and the ROS level was assessed using DCFH-DA.

The green tea derivative (-)-epigallocatechin-3-gallate (EGCG), a major polyphenolic compound found in green tea, has been shown to possess potent antioxidant properties²⁵. We added EGCG (10 µM) to high-glucose medium and found that EGCG significantly inhibited the increase in ROS induced by high glucose in LECs (Fig. 6B). To verify whether the antioxidant EGCG could reverse oxidative stress-induced LECs apoptosis in the HG environment, western blot experiments were performed. We found that EGCG increased the expression level of the MCL1 protein while decreasing the expression of apoptosis-related proteins such as cleaved-caspase3 and PARP in LECs in the HG environment (Fig. 7A, B and C). The above results suggest that, as an antioxidant, EGCG can inhibit high glucose-induced LECs apoptosis by inhibiting ROS.

Fig. 7.

EGCG regulates the ROS-USP10-MCL1 pathway. (A) HLE-B3 and SRA01/04 cells were treated with EGCG (10 µM), glucose (50 mM) or EGCG (10 µM)+glucose (50 mM) for 72 h. The protein expression of MCL1, Caspase3, PARP and GAPDH was assessed by western blot assays. GAPDH was used as a reference protein. (B) Relative level of MCL1 adjusted to the level of GAPDH (ns: not statistically significant, *P < 0.05, **P < 0.01). (C) Relative level of cleaved-PARP adjusted to the level of GAPDH (ns: not statistically significant, *P < 0.05).

EGCG delayed the progression of DC

To confirm the effect of the antioxidant EGCG on DC, we established a diabetic rat model in which the rats were treated with an aqueous EGCG solution (Fig. 8A). We found that EGCG significantly delayed the progression of cataracts in diabetic rats after 9 weeks (Fig. 8B). The immunohistochemical results also revealed that the protein expression of USP10 and MCL1 in the LECs of diabetic rats was significantly downregulated, whereas the expression levels in the LECs of rats treated with EGCG were reversed.

Fig. 8.

EGCG delays the progression of diabetic cataract. (A) Flowchart for the animal experiment. (By Figdraw, ID: IURTP6ccb4) (B) Anterior segment photos of the control group, STZ (65 mg/kg)-treated group, and STZ (65 mg/kg)+EGCG (200 mg/kg·d)-treated group rats at 0, 3, 6, and 9 weeks after successful modelling. Representative images of IHC staining for USP10 (C) and MCL1 (D) in lens tissues from the rats in NC, STZ and STZ+EGCG groups. (magnification: eye ball: ×1.2, Lens epithelial cells: ×14, antibodies uesd: anti-USP10 (D7A5, 1:100) and anti-MCL1 (66026-1-Ig, 1:400))

Discussion

DC is the most common complication of diabetes and is one of the leading causes of blindness worldwide²⁶. The molecular mechanisms underlying DC progression require further clarification for the development of more effective therapeutic approaches.

According to previous studies, HG readily induces LECs apoptosis. To explore the underlying mechanism, we cultured LECs in HG medium to simulate diabetes in vitro. Under HG conditions, the survival rate of LECs significantly decreased, while the expression levels of proapoptotic proteins, such as cleaved-caspase9, cleaved-caspase3 and PARP, increased. PI staining also revealed that HG promoted apoptosis in cells.

In addition, we found that the expression of the antiapoptotic protein MCL1 in cells was downregulated in the HG environment. To validate the regulatory effect of MCL1 on LECs apoptosis, we overexpressed MCL1 in LECs and found that MCL1 overexpression significantly attenuated HG-induced apoptosis. These results suggest that MCL1 plays an important role in the HG-induced apoptosis of LECs.

The stability of the MCL1 protein is affected by posttranslational modifications such as ubiquitination, phosphorylation, and acetylation¹¹. To explore whether MCL1 expression is affected by ubiquitination in HG-treated cells, we overexpressed the UB plasmid in LECs. Co-IP assays revealed that the ubiquitination level of MCL1 increased in the HG environment, a finding that confirmed our hypothesis.

USP10 is a deubiquitination enzyme that reportedly regulates the stability and activity of some apoptosis associated proteins. We found that HG induced the downregulation of USP10 expression. To verify whether USP10 is an upstream regulatory factor of MCL1, we overexpressed USP10 in LECs, and the results showed that USP10 reversed the HG-induced downregulation of MCL1 expression. Furthermore, the co-IP results indicated that there is a protein interaction between USP10 and MCL1. In addition, the HG environment inhibited the binding of USP10 to the MCL1 protein. Taken together, our preliminary results revealed that MCL1 is a potential USP10 substrate in LECs. The HG environment weakened the binding of USP10 to MCL1, thereby reducing the level of MCL1 deubiquitination and ultimately inducing the degradation of MCL1 and the apoptosis of LECs.

As a USP family deubiquitinating ligase, USP10 consists of a relatively large N-terminal region, a USP catalytic structural domain and a relatively small C-terminal region^[3]. However, the mechanism of MCL1 deubiquitination by USP10 was not clarified in our study. The specific binding sites of USP10 and MCL1 requires further investigation.

Oxidative stress plays a significant role in the development of DC. EGCG is a major polyphenolic compound found in green tea that has been shown to possess potent antioxidant properties. In our study, we found that EGCG significantly inhibited the increase in ROS induced by HG in LECs and increased the expression level of the MCL1 protein while decreasing the expression of apoptosis-related proteins such as cleaved-caspase3 and PARP in LECs in the HG environment, which suggests that EGCG inhibits HG-induced LEC apoptosis by decreasing ROS levels.

Previous studies have shown that USP10 can inhibit oxidative stress through multiple molecular pathways^27,28. As a member of the cysteine protease family, USP10 can also be regulated by ROS during oxidative stress, adding to the complexity of the mechanisms by which DUBs may be regulated²⁹. In our study, both ROS and USP10 had regulatory effects on the expression level of MCL1. However, further experimental confirmation is needed to validate the regulatory relationship between USP10 and ROS in DC.

Despite these interesting findings, our research is limited mainly to the cellular and molecular levels. Whether EGCG retards DC through ROS and USP10 in animal models needs to be verified. Therefore, our future experiments will aim to address these important issues.

In conclusion, MCL1 delays the progression of DC by inhibiting the apoptosis of LECs. The deubiquitinase USP10 can deubiquitinate and stabilize MCL1 to prevent its degradation in the HG environment. Moreover, the antioxidant EGCG significantly delays cataract progression in diabetic rats by reversing oxidative stress-induced LECs apoptosis in the HG environment. Our study contributes to elucidating the molecular mechanism of DC and provides new therapeutic targets and ideas for the subsequent development of nonsurgical treatment options.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

YQ .J and WJ.W have made contributions to Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft.HR.X has made contributions to Formal analysis, Software, Validation, Investigation.H.P has made contributions to Resources, Software, Data curation, Investigation, Formal analysis.YX.L has made contributions to Software, Data curation, Investigation, Formal analysis.H.Z has made contributions to Funding acquisition, Project administration, Resources, Supervision, Writing –review & editing, Conceptualization.XF.Z has made contributions to Conceptualization, Formal analysis, Methodology, Project administration, Supervision, Writing –review & editing.

Funding

The present study was supported by the National Natural Science Foundation of China (no. 81873677) and the Shandong Natural Science Foundation (no. ZR2020QH147).

Data availability

The datasets supporting the conclusions of this article are included within the article.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval and consent to participate

This study was approved by the Animal Experimental Ethics Committee of the Shandong Provincial Hospital Affiliated with Shandong First Medical University (approval number: No. 2023 − 180).