| Objective:Diabetes is one of diabetic chronic complications of hearing impairment. Hearing damage caused by diabetes pathogenesis is not clear. Cochlear high metabolic characteristics and the ability to produce free radicals make it vulnerable to reactive oxygen species (ROS) oxidative damage. Existing research shows that reactive oxygen species (ROS) can activate a variety of the irreversibility of cochlear cells apoptosis signaling pathways damage, is a key factor in occurrence and development of sensorineural deafness. Diabetic deafness is also a kind of sensorineural deafness. A large number of audiological testing found that people with diabetes hair cells degeneration appeared. Oxidative stress caused by long-term high blood sugar and its metabolites is key factor inducing diabetic deafness. Thioredoxin (Trx) is one of the main antioxidants in the body, and the Trx in reduce apoptosis and promote cell proliferation/survival play a key role. Sulforaphane (SF) is widely exist in broccoli and other cruciferous vegetables and medicinal plants such as indirect antioxidants of semen raphani, through the induction of type II detoxification enzymes, inhibit the I phase metabolic enzymes play its antioxidant function. SF, can through the Trx and expression of TrxR protective and confirmed by many experiments.NF-E2-related factor 2 (Nrf2) is the core of the influence ARE mediated gene transcription protein, combined with all area after adjusting the phase ? metabolism enzyme gene transcription. Silk crack the original amp-activated protein kinase (MAPK) is one of the key signaling systems biology, involved in mediating multiple cellular processes.This topic through the establishment of type 2 diabetes in mice model, discusses sulforaphane (SF) diabetes caused by cochlear degenerative change of protection mechanism, in order to provide clinical prevention and treatment of diabetic deafness new experimental theoretical basis and guidance.Methods:Balb/c mice can be divided into type 2 diabetes mellitus (T2DM) and normal group (control). First of all, according to the internationally recognized modeling standards, type 2 diabetes animal model to establish animal model of type 2 diabetes, Then with different doses of SF (0.5 mg/kg,1 mg/kg,2 mg/kg) and the same dose of SF, different delivery time (five days, ten days, fifteen days) respectively deal with T2DM group of mice to determine the best dosage and suitable for T2DM duration and observation group and Control group mice cochlea Trx gene and protein expression levels in the organization. With SF, and finally the Trx inhibitors PX-12 in the Control group and T2DM parallel processing, carried out in two groups were divided into Control group and treatment group (SF), and the experimental group (SF+PX-12). We through the cochlear succinate dehydrogenase (succinic dehydrogenase, SDH) dyeing cochlear hair cell count and cochlear slice HE analyzes cochlear tissue morphology; Adopt the method of cochlear paraffin immunofluorescence histochemical detection TRX with ASK1 in location and expression of cochlear tissue; The Real-time PCR detection Trx in cochlea organization and Txnip genes expression and using Western blot method to detect Trx mouse cochlear tissue and transcription factor Nrf2, p-Erk, and apoptosis regulation protein kinase ASK1 protein expression level.Results:1. The different dosage of SF, and at the same dose of different duration of treatment for T2DM group mice cochlea in Trx protein and gene levels in the organization.First with different doses of SF (0.5 mg/kg,1 mg/kg,2 mg/kg) mice after dealing with T2DM group, Western blot detection Trx protein levels, according to the results of SF, each dose treatment groups compared with T2DM group increased significantly (p< 0.05,p<0.01,p<0.05). Compared with normal group,1 mg/kg,2 mg/kg dose group of Trx have different degree (p<0.01,p<0.05), of which 1.0 mg/kg dose group of the most significant. Real-time PCR detection results show that with Western blot test results consistent, each dose group of Trx gene level in the cochlea organization has raised in the T2DM untreated group,1 mg/kg dose groups in the most significant (p< 0.01), compared with normal control group was statistically difference (p<0.05).Then use SF1 mg/kg dose in different time (five days, ten days, fifteen days) with T2DM group of mice after Real-time PCR detection and Western blot test results found that the expression level of cochlear Trx in T2DM group mice and drug use is related to time, of which the most suitable for 15 days (p<0.01).2. SF, and inhibitors of Trx PX-12 on the mechanism of action of T2DM degeneratie cochlear hair cells in mice.2.1 cochlear succinic dehydrogenase(SDH) staining to observe the T2DM group compared with normal group lower bottom back especially hair arrangement is irregular, SDH dyeing becomes shallow. Back from mouse cochlear top back to bottom hair cells loss counting mean equal distance, according to the results of the T2DM group compared with normal group of outer hair cells mean reduced to some extent and inner hair cells mean reduced slightly. The T2DM group compared with normal group of outer hair cells mean significantly reduced (p<0.001) and inner hair cells mean decreased (p<0.05). In T2DM group simply give the SF group of outer hair cells compared with T2DM controls mean significantly increased (p<0.001) and inner hair cells mean increased (p<0.05). SF in T2DM group and the group by the joint action of PX-12 compared with pure give SF group outer hair cells mean has decreased (p< 0.01) and inner hair cells mean decreased (p<0.05). From on macroscopic observation cochlear organization HE dyeing slice missing hair cells according to the results and hair cells SDH count mean trend is consistent.2.2 cochlear paraffin section immunity fluorescence was noted.immunohistochemical SF, and inhibitors of TRX PX-12 in T2DM cochlear tissue in mice (red fluorescence) and ASK1 (green fluorescence).expression TRX with ASK1 expression mainly in the cochlear hair cells in mice, the T2DM group comparison cochlear hair cells in mice and normal mice TRX expression decreased significantly, while the expression of ASK1 significantly enhanced. In T2DM group simply give the SF group compared with T2DM group cochlear hair cells in the TRX expression enhanced obviously, the expression of ASK1 decreased significantly. SF in T2DM group and the group by the joint action of PX-12 compared with pure give SF group cochlear hair cells in the expression of TRX has subsided, the expression of ASK 1 increased.2.3 SF, and inhibitors of Trx PX-12 TRX in T2DM mice cochlea and ASK1 Nrf2 and P-Erk expressionUse the method of Real-time PCR testing groups of cochlear Trx and Txnip gene expression levels in the organization, and test results show that compared with normal control group, mice cochlea organization the Trx in T2DM group was obviously reduced (P<0.01), the expression of Txnip significantly raised (P<0.01). Simply give the SF, dealing with the normal group of mice compared with normal control group a Trx gene expression in the cochlea raised (P<0.05), the expression of Txnip is lower (P <0.05). SF, and PX-12 joint processing in normal mice and simply give SF, processing more Trx gene expression is lower in normal mice (P<0.05), the expression of Txnip increase (P<0.05). Simple to SF, treatment of T2DM group mice compared with T2DM controls a Trx gene expression in the cochlea raised (P<0.05), the expression of Txnip is lower (P<0.05). The SF, and PX-12 joint treatment of T2DM group mice compared with pure to SF, treatment of T2DM group mice Trx gene expression is lower (P<0.05), the expression of Txnip increase (P<0.05). Western blot test results show that compared with normal control group, Trx in T2DM group mouse cochlear tissue, Nrf2 and P-Erk protein expression levels significantly lowered (P<0.01,P<0.05,P<0.0, 5), the expression of ASK1 significantly raised (P<0.01).Simply give the SF processing in normal mice and normal control group to compare the Trx in the cochlea, Nrf2 and P-Erk protein expression increase (P<0.05,P<0.05,P<0.0,5), the expression of ASK1 is lower (P<0.01). SF, and PX-12 joint processing in normal mice and simply give more Trx in SF, processing of normal mice, Nrf2 and P-Erk expression have different degrees of cut (P<0.01, P<0.05, P<0.0,5), the expression of ASK1 increase (P<0.05). Simply give the SF, treatment of T2DM group of mice with T2DM control group to compare the Trx in the cochlea, Nrf2 and P-Erk expression have varying degrees of increase (P<0.05, P<0.05,P<0.0,1), the expression of ASK1 is lower (P<0.05). The SF, and PX-12 joint treatment of T2DM group mice compared with pure to SF, treatment of T2DM group mice Trx, Nrf2 and P-Erk expression have different degrees of cut (P<0.05, P<0.01, P<0.0,5), the expression of ASK.1 increase (P< 0.05). That SF, by acting on P-Erk/Nrf2 oxidation raised Trx expression of the signal transduction pathway, thereby inhibiting the ASK1 causes the activation of signaling pathways of apoptosis.Conclusion:1. Diabetes mice significantly, weakened Trx expression in cells and Txnip ASK1 expression. End of cochlear outer hair cells loss back2. SF can effectively raise the Trx of cochlear hair cells in inhibition to Txnip and ASK1 mediated activation of downstream apoptotic pathways, the degeneration of cochlear hair cells in diabetes mice protection.3. P-Erk/Nrf2 antioxidant polymerase chain associated with the increase of SF, mediated Trx expression. |
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