Up-regulation thioredoxin contributes to inhibite diabetic hearing impairment

Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia. An association between DM and hearing impairment has been widely discussed. It’s essential to nd effective prevetion or diagnosis of diabetic hearing loss. Thioredoxin (Trx) is a small molecule protein (12kDa) and plays biological functions such as anti-apoptotic, transcriptional regulation. In this study, our aim is to clarify the protective effect of Trx on diabetic hearing loss to nd the early potential therapeutic target of diabetic hearing impairment in clinic in the future.


Introduction
Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia. The number of patients with DM is increasing each year and there will be a variety of complications such as diabetic retinopathy (DR)[1], nervous system damage[2], kidney damage[3] and cardiovascular disease [4] with the progression of diabetes. Besides, an association between DM and hearing impairment has been widely discussed. The pathogenesis of diabetic hearing loss remains unclear [5,6] and affected the life of diabetes patients. Thus, it is strongly necessary to understand the mechanisms and nd some ways to inhibite diabetic heraing impairment. There are many factors participated in this process such as oxidative stress [7], endoplasmic reticulum stress [8] (ERS), and autophagy [9]etc.
Endoplasmic reticulum is involved in many important physiological functions in eukaryotic cells such as post-translational modi cation, folding, assembly, lipid synthesis and intracellular calcium storage [10]. Exposure to high glucose for a long time may lead to protein synthesis disorders or accumulation of misfolded proteins in endoplasmic reticulum lumen, which may lead to ERS [11]. Glucose-regulated protein 78 (GRP78) plays an important role in regulating ERS [12] .
Autophagy is a highly conservative catabolic process in eukaryotic cells, which plays a crucial role in maintaining normal life activities. It has been reported that autophagy induction can be against in ammation [13], improve senescence [14], and prevent neurodegeneration [15]. As a protective mechanism of cells in an adverse environment, with the development of diabetes, autophagy disorders lead to autophagic apoptosis [16].
Thioredoxin (Trx) is a small redox protein existing in tissues which contains a dithiol-disul de active site: -Cys-Gly-Pro-Cys- [17]. It can remove intracellular ROS and protect cell from oxidative damage effectively [18]. Trx is a vital regulator of apoptotic signal-regulating kinase 1(ASK1) functions. When Trx is oxidized, ASK1 is dissociated from Trx and interacts with TRAF2/6 to form ASK1-TRAF2-IRE1α (ERS marker) complex which ultimately promote cell apoptosis and death [19,20]. Moreover, we also found that Trx can regulate autophagy through thioredoxin-interacting protein (Txnip)-mTOR pathway [21]. Our former researches indicated that Sulforaphane (SF) which is widely found in cruciferous plants, including cabbage, broccoli and radish also can up-regulate Trx expression [22] to delay photoreceptor degeneration in diabetes [23].
Basing on these evidences, in this study we will up-regulate Trx expression through SF treatment or transgenetic method to explore the protective effect of Trx on diabetes-indueced cochlear hair cell degeneration and related mechanism aiming to provide some evidence for diabetic hairing impairment prevention or treatment in clinic in the future.

Patients
A total of 45 patients with diabetes in the Endocrinology Department of the Second A liated Hospital of Dalian Medical University. According to the average value of pure tone audiometry from 0.5kH-4kHz, the patients were divided into two groups: Diabetics with normal hearing (DM1, 20 cases, 40 ears) and diabetics with abnormal hearing (DM2, 25 cases, 50 ears). People with healthy examination were selected as the normal control group (NC, 25 cases, 50 ears). Signed consent was obtained from all patients.

Otoacoustic emissions
OAEs are sounds caused by the motion of the cochlea's sensory hair cells as they energetically respond to auditory stimulation. The method of OAEs recording used in this study were the distortion product otoacoustic emissions(DPOAEs) and transient evoked otoacoustic emissions (TEOAEs). The instruments were GSI-61 dual channel diagnostic audiometer. Automatic tympanometer of GSI-39version and Cochlear Emission Analyzer of Capella Denmark GN Audiometrics A/S company. The frequencies analyzed in this study were 0.5 k, 1 k, 2k, 4 k and 8 kHz.

Animal Care
Male Trx transgenic (Tg) mice (C57BL/6J background, six-week-old, 20±2g) were obtained from Dalian Medical University Laboratory Animal Center. All experiments involving animals in this study were executed in keeping with institutional guidelines for the care and use of laboratory animals, the experimental program was approved by the institutional Animal Care and Use committees of Dalian Medical University Laboratory Animal Center. All experimental animals were fed adaptively (immobile temperature 22±2℃, su cient water and food, 12 hours light / dark: average illuminance of 80 lx). Genomic DNA from mouse tail was ampli ed by PCR using the following sets of primers: HTXN-380F 5'-CAGTACATCAATGGGCGTGG-3', HTXN-956R 5'-CGCGGTACCGTCGACTTAGA-3'. The conditions of PCR ampli cation for synthesized Trx were as follows: 95℃ for 30 seconds, 95℃ for 10 seconds, 65℃ for 30 seconds, 72℃ for 1 minutes and for 35 cycles, 72℃ for 10 minutes, 4℃ for endless. The PCR products were running on a 1% agarose gel at 80 V for 40 minutes and stained with ethidium bromide (EB). The PCR product of Tg-Trx is 577 bp.The data was analyzed with a GDS-8000 Bio imaging system (UVP, Upland, CA).

Diabetes model establishment
After adaptive feeding, the diabetic model was inducted by intraperitoneal injected streptozotozin(50mg/kg) continued for 5 days and control group was given intraperitoneal injection of citrate buffer. Diabetes was de ned as follow: random blood-glucose ≥16.7 mmol/L [24].

Drug treatment
The diabetic mice were intraperitoneal injection with 1.0 mg/kg SF (S8046, LKT Laboratories Inc., St. Paul, MN, USA) and/or 1.0 mg/kg PX12 (Trx inhibitor) for 2 weeks. The control mice were injected with saline solution. Animals were sacri ced by CO 2 asphyxiation after treatment. .

Hair cell counting
Animals were sacri ced with overdose CO 2 and the cochleae were quickly removed from the skull and put them into the NBT (Solarbio ® , N8140 ) working uid (1:1:2, 0.2 M sodium succinate, 0.2 M PBS pH 7.6, 0.1% tetranitro-blue tetrazolium). The round and oval windows were removed and the apex of the cochlea was opened to facilitate perfusion. NBT working uid is slowly injected into the cochlea along the circular window. The working uid owed from the hole at the top of the cochlea, the perfusion was successful.
The cochlea was placed in NBT working uid, incubated at 37℃ for 1 hour, and xed with 10% formalin for 2 days. 7% EDTA decalci ed solution at room temperature until cochlear osteomalacia. The hair cells were counted from the top to the bottom of the cochlea at the same distance (200 µm) under a light microscope. The hair cells numbers were compared to the normal numbers obtained in the group and cell losses were expressed as pecentage. Cell losses were plotted as a function of cochlear length (cochleogram).

Immuno uorescence staining
The mice were euthanized with excessive CO 2 and their cochlear were removed. The cochlear were xed in 10% formaldehyde solution for 2 days after removing the auditory vesicles. 7% EDTA decalci ed solution at room temperature until cochlear osteomalacia. After dehydration in alcohol, the cochlear were cut into 5-μm paraff in sections that parallel to the axis of the cochlea. Primary antibodies were anti-Trx (Proteintech ® , 14999-1-AP, 1:200), anti-ASK1 (Santa Cruz, sc-5294, 1:200). Antigens were visualized after incubation with Alexa Fluor® 488 or 649-conjugated goat IgGs (Molecular Probes). Fluorescent images were captured using microscope (Leica DM6000B uorescence microscope, Germany).

Statistical analysis
The data were presented using mean±SD and the differences among groups were determined via oneway analysis of variance and t test. All performs were made by GraphPad Prism software (Ver.5.0). A p value < 0.05 was considered signi cant.

Results
Diabetes induced hearing impairment in pateints.
As show in Fig. 1 A, the average PTA value of DM1 and the normal group are within the normal range, there is no signi cant difference between the PTA value of DM1 and normal group in 0.25kHz and 0.5kHz, while it's signi cantly higher in DM1 than control group in 1k-8kHz. And the PTA value of DM2 in frequency 0.5k-8kHz is signi cantly increased compared with DM. The OAE results which including DPOAE level, S/N-ratio and TEOAE correlaton in DM1 and DM2 are all signi cantly increased compared with control group. Simultaneously, they are signi cantly decreased in DM2 compared with DM1( Fig. 1B-D). The auditory testing results indicate the diabetic cochlea have been damaged before the hearing change, the early hearing loss in diabetics are mainly in high frequencies and involve full frequency gradually.
Diabetes induced cochlear hair cell degenerationin vivo.
As shown in Fig.2A&B, as the progression of diabetes, the FBG and homeostasis model assessmentinsulin resistance index (HOMA-IR) were increased signi cantly in diabetic group compared with control group. The cochlea from diabetic mice were isolated for SDH staining to observe the degeneration of the outer hair cell (OHC) and inner hair cell (IHC). Fig.2 C-E present OHC loss (D) and IHC loss (E) in diabetic mice as a function of cochlear length (cochleogram). The degeneration started from the basal turn and expanded progressively with time towards the apical turn. At 10 day, both OHC loss and IHC loss occurred. The percentage of loss was increasing with the diabetes process. However, IHC degeneration appeared to develop more slowly than the OHC. There was no hair cell loss observed in control group.
We also used western blot to detect the expression of Trx. The results showed that the relative expression of Trx was decreased gradully and was positively correlated with the loss of cochlear hair cells in diabetic mice (Fig.1 F&G).
Up-regulated Trx expression by SF inhibited diabetic induced cochlear hair cell injury.
As show in Fig.3 A&B, Trx expression was signi cantly up-regulated after treated with SF. The total loss ratio of OHC/IHC at 31.8%/16.3% in diabetic group compared with WT group. However, the loss ratio of OHC/IHC(26.5%/13.8%) was inhibited in diabetic mice after SF treatment compared with diabetic mice. The process could be inhibited by PX12 (Fig.3 C&D&E). we also detect the expression of Trx and Txnip. The results showed that the expression of Trx and Txnip was decreased or increased signi cantly in diabetic group compared with WT group, however, the expression of Trx and Txnip was up/downregulated after SF treatment in diabetic group which can be reversed by PX-12 (Fig.3 F&G).
Up-regulated Trx expression by SF inhibited diabetic induced cochlear hair cell injury through downregulating ASK1 and Txnip.
As shown in Fig.4 A, Trx (red) and ASK1 (green) were mainly located at the OHC/IHC of cochlea, the red uorescence of Trx in the diabetic group was signi cantly decreased, while the green uorescence of ASK1 was signi cantly increased compared with the control group, which suggesting that diabetes can down-regulate Trx expression and up-regulate ASK1 expression in cochlea. After SF treatment, the red uorescence was increased and the green uorescence was signi cantly decreased compared with the diabetic group. However, the green uorescence (ASK1) was signi cantly increased and the red uorescence (Trx) was signi cantly decreased after SF and PX12 treatment. We also used western blot to detect the expression of Trx and ASK1 (Fig.4 B-D).
Over-expressed Trx can delay diabetes induced cochlear hair cell degenerationin vivo.
As shown in Fig.5 A, over-expressed Trx transgenic mice were identi ed and randomly divided into Tg(-)-DM group and Tg(+)-DM group. As show in Fig.5 B, FBG in DM group was up to the standard of diabetes. Subsequently, we counted the hair cell under microscope with SDH staining to observe the loss of cochlear hair cells. The results demonstrated that OHC/IHC loss was signi cantly delayed in Tg(+)-DM group compared with Tg(-)-DM group (Fig.5 C&D). Moreover, we used qRT-PCR to measure gene expression of Tfam, ASK1 and Txnip. As shown in Fig.5 E-G, the expression of Tfam was increased and the expression of ASK1 and Txnip was decreased in Tg(+)-DM group compared with Tg(-)-DM group.
Over-expressed Trx can delay diabetes induced cochlear hair cell degeneration through regulating mitochondrial pathway/ endoplasmic reticulum stress (ERS) / autophagy in vivo.
We used western blot to detect protein expression for mitochondrial pathway, ERS and autophagy. As shown in Fig.6 A-D, the expression of PGC1α was decreased in the Tg(-)-DM group compared with Tg(-) group, while it was increased in the Tg(+)-DM group (Fig. 6 B). The expression of pro-apoptotic protein Bax in Tg(+)-DM group was signi cantly decreased compared with Tg(-)-DM group (Fig. 6 C). Moreover, the expression of bcl-2 expression in Tg(-)-DM group was signi cantly decreased compared with Tg(-) group, however, it was increased in Tg(+) group compared with Tg(-) group (Fig. 6 D). As shown in Fig.6 E-H, the expression of ASK1, GRP78 and CHOP in Tg(-)-DM group was signi cantly increased compared with Tg(-) group , while the expression of ASK1, GRP78 and CHOP was signi cantly decreased in the Tg(+)-DM group. As shown in Fig.6 I-L, the expression of Txnip signi cantly increased in Tg(-)-DM group, which it was reversed by over-expression Trx. The expression of LC3-II was decreased in the Tg(-)-DM group, while the expression in the Tg(+)-DM group was increased than Tg(-)-DM group (Fig.6 K). Besides, the expression of p62 was increased in Tg(-)-DM group, while it was decreased in Tg(+) group compared with the Tg(-) group (Fig. 6 L). These results was summarized in Fig.7.

Discussion
Diabetes induced hearing impairment is one of the serious complications of DM. Because it's high incidence, it was attracted more and more people's attention. The onset of diabetic hearing impairment is hidden and the course of the disease is slow, which seriously affects the life quality of patients. Most patients have irreversible hearing impairment [25]. In our research, we found 84% of diabetics in DM2 had bilateral hearing loss. We also found cochlear impairment in diabetes patients with normal PTA by otoacoustic emission (OAE). Apply easily, possible without cooperation, and not taking a long time are main features of the test. The sensitivity of TEOAE is higher in detection, while DPOAE has been used more frequently in clinic, because it enables the detection of frequencies in wider intervals in the early detection of cochlear disorders [26]. The frequencies analyzed in this study were 0.5k, 1k, 2k, 4k and 8kHz. The value of DPOAE level records was lower with the frequencies increasing in diabetes pateints compared with the healthy people.
With the development of diabetes, permanent hearing loss or imbalance is caused by hair cell loss, internal auditory arteriosclerosis and delayed auditory brainstem response (ABR) delayed wave V [27]. Therefore, illuminating the relationship between diabetes and hearing impairment and its pathogenesis is of great signi cance for early diagnosis, prevention and treatment. Mitochondrial damage, endoplasmic reticulum stress and autophagy disorder caused by persistent hyperglycemia in diabetes plays an important role in cochlear hair cell damage and hearing loss [28,29,30].
Along with glutathione, thioredoxin (Trx), a small 12kDa protein widely found in organism, is one of the two main antioxidants in mammals [31]. Moreover, Trx plays a pivotal role in maintaining the intracellular environment and regulating prolifertion and transcriptional [32]. Our previous study also found that the crucial biological functions of Trx on anti-oxidation and anti-apoptosis in cells, which is important for maintaining cell metabolism [33,34].
In this study, up-regulating Trx by SF and transgenic mice (Trx) were used to explore the role of Trx in the process of diabetes-induced cochlear hair cell damage and hearing loss in mice. We demonstrated that there was a signi cant correlation between Trx expression decreasing and cochlear hair cell degeneration in diabetic mice. Moreover, we also found that up-regulating Trx by SF could protect the hair cells loss induced by diabetes. Furthermore, the expression of ASK1 can by inhibited after Trx expression increasing which was detected in the cochlear hair cells (Fig.4). Besides, in our former research reported that Grape Seed Proanthocyanidin Extract (GSPE) can prevent retinal degeneration in diabetic mice by mediating upregulation Trx and reducing ER stress [35]. These results indicated up-regulating Trx would be a potential way to delay neurodegeneration induced by diabetes. After over-expression Trx in mice, we further explored the protective effect and mechanism of Trx on the diabetes-induced cochlear hair cells. We found that the outer hair cells were arranged loosely and irregularly in Tg(-)-DM, while the volume of hair cells was increased and arranged regularly in Tg(+)-DM group compared with Tg(-)-DM group.
In our research, we found that mitochondrial pathway, ERS and autophagy involved in process of cochlear hair cell damage. In Tg(+)-DM group the expression of PGC1α and Bcl-2 was increased and the expression of Bax was decreased compared with Tg(-)-DM. It suggested that Trx can effectively improve the loss of cochlear hair cells in diabetes through regulating mitochondrial pathway. Scholars also have proved that Trx over-expression extends antioxidant protection, attenuates mitochondrial damage, and activates mitochondrial turnover such as mitophagy and biogenesis [36].
Endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions. It is essential for cell homeostasis, development and stress response [10]. And it can be activated by many unfavorable factors. Studies have shown that high glucose stimulation can lead to the occurrence of ER stress response [37]. When ER stress is activated, several apoptotic signaling pathways can be activated to induce cell apoptosis. Trx can alleviate the neurodegeneration of the disease by inhibiting the endoplasmic reticulum stress in MPP + environment [17]. Our work also con rmed that the expression of ER stress marker protein GRP78 in DM group was signi cantly increased compared with non-diabetic group. In transgenic Trx diabetic mice with down-regulating the expression of ASK1 leading to the expression of GRP78 and CHOP was decreased in cochlear compared with Tg(-)-DM mice. It indicates that diabetes can signi cantly activate ER stress. Trx can suppress ER stress via ASK1 to protect hair cells from diabetes.
Autophagy is also a highly conserved lysosome degradation pathway in eukaryotes, responsible for providing metabolic energy, circulating intracellular components, and eliminating harmful molecules, organelles and abnormal accumulation of substances [38]. Studies have shown that autophagy is essential for the development and maturation of the inner ear of vertebrates, which is the basic intracellular pathway [30]. Autophagy is essential in neurons and the postnatal onset of hearing and the concomitant increase in neuronal activity is correlated with the induction of autophagy in the cochlea [39,30]. After over-expression of Trx with down-regulating Txnip expression, the expression of LC3-II, an autophagy marker protein, was up-regulated in Tg(+)-DM, suggesting that Trx can activate autophagy and increase the encapsulation of products to be degraded in cochlear cells. Trx can reduce the expression of autophagic lysosome degradation protein p62 and promote autophagic lysosome degradation which accelerated the autophagy ux.
In summary, up-regulate Trx by SF and over-expression of Trx in mice can protect the cochlea hair cell damage induced by diabetes. The underlying mechanism may be related to regulate ER stress through ASK1 and autophagy or mitochondria pathway through Txnip.

Conclusion
Up-regulating the expression of Trx can delay diabetes-induced cochlear hair cells degeneration and the underlying mechanism may be related to regulate ER stress through ASK1 and autophagy or mitochondria pathway through Txnip.

Declarations
Ethics approval and consent to participate All animal experiments followed the institutional Animal Care and Use committees of Dalian Medical University Laboratory Animal Center.

Consent to publication
All authors read and approve of the nal manuscript for publication.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Author's contribution Xiang Ren, Jinjuan Lv, Yuzhen Fu and Na Zhang designed, performed the experiments, and evaluated the data. Yuzhen Fu, Na Zhang, Chenghong Zhang, Zhenghao Dong, Maryam Chudhary and Shiwen Zhong contributed to reagents, materials and analytical tools and provided records and interpretation of the data. Xiang Ren, Jinjuan Lv and Na Zhang contributed to manuscript preparation. Li Kong and Hui Kong were the guarantor for this work and had full access to all the data in the work and takes responsibility for the integrity of the data and the accuracy of the data analysis.    The protective effect of Trx on cochlear hair cell in diabetic mice. Trx over-expression mice were identi ed(A) and RBG was uesd to evaluate diabetic model(B). Counting the outer cochlear hair cells (C) and inner cochlear hair cells(D) in diabetic mice. Q-PCR were used to evaluate the expression of Tfam (E), ASK1 (F) and Txnip (G) in diabetic mice. The data are expressed as the mean ± SD (n=3 for each group). *p<0.05, **p<0.01, ***p<0.01.

Figure 6
The underlying protective mechanism of Trx on cochlear hair cells in diabetes. Western blot was used to detected the experssion of mitochondrial related proteins (  Summarize the protective effect and related mechanism of Trx on diabetes-induced cochlea hair cell damage.