| Objective: To analyze the differences in the expression of Nrf2 protein and TLRs in the ear mucosal tissues of different types of otitis media and the correlation between the expression levels of Nrf2 and TLRs in chronic suppurative otitis media.Explain the relationship between the expression of Nrf2 in the mucosal tissue of the middle ear in chronic suppurative otitis media and the pathogenesis of chronic suppurative otitis media.To explore the effect of Nrf2 and TLR4 expression patterns on the pro-inflammatory cytokines in the middle ear mucosal tissue of chronic suppurative otitis media.On this basis,we will further explore the internal regulation mechanism of Nrf2 regulating TLR4 axis in the pathogenesis of chronic suppurative otitis media,and lay a scientific foundation for revealing the pathogenesis of chronic suppurative otitis media and the underlying molecular mechanism of acute otitis media to chronic suppurative otitis media.Methods: Part I:(1)The expression levels of Nrf2 and TLRs(TLR2,TLR4,TLR5 and TLR9)in the middle ear tissues of patients with chronic suppurative otitis media(CSOM),diseases otitis media(DOM)and non-otitis media(Non-OM)were detected by RT-q PCR method.(2)In order to verify the accuracy of the RT-q PCR results,Western blot was used to detect the differences in the expression levels of Nrf2 and TLRs in the middle ear tissues of the above three types of otitis media.(3)Persson correlation analysis method was used to analyze the correlation and interaction between the expression levels of Nrf2 and TLR4 in chronic suppurative otitis media.Part II:(1)The differential expression of pro-inflammatory cytokines(TNF-α,IL-1β,IFN-γ and IL-6)in the middle ear tissues of CSOM,DOM,Non-OM were detected by RT-q PCR.(2)The accuracy of RT-q PCR results was verified by ELISA method.To analyze the significance of the expression levels of chronic inflammation in CSOM,DOM,and Non-OM.Part III:(1)The high-dose lipopolysaccharide(LPS)-induced AOM and continuous low-dose LPS-induced CSOM models in mice were established.Western blot was used to detect the differences in the levels of pro-proliferation-related proteins(Cyclin D1 and CDK2)and pro-apoptotic proteins(cracked Caspase-3 and Bax)in the middle ear tissues of the three groups of AOM,CSOM and Control.(2)RT-q PCR was used to detect the differences in the expression levels of pro-inflammatory cytokines in the middle ear tissues of the three groups of AOM,CSOM,and Control;The accuracy of RT-q PCR results was verified by ELISA method.(3)RT-q PCR was used to detect the differences in the expression levels of Nrf2 and TLR4 in the middle ear tissues of the AOM,CSOM,and Control groups;Western blot was used to verify the accuracy of the above RT-q PCR results.(4)To verify the knockdown efficiency of Nrf2 and the overexpression efficiency of TLR4,RT-q PCR was used to detect the difference in the expression level of Nrf2 in CSOM,COSM+KD-Nrf2 middle ear tissues and TLR4 in CSOM,COSM+OE-TLR4 middle ear tissues.(5)The difference of TLR4 expression levels in middle ear tissue of CSOM,COSM KD-Nrf2,Control were detected by RT-q PCR method;Western blot method was used to verify the accuracy of the above RT-q PCR results.RT-q PCR method was used to detect the difference of Nrf2 expression levels in the middle ear tissues of CSOM,COSM+OE-NTLR4,Control;Western blot method was used to verify the accuracy of the above RT-q PCR results.(6)The difference of proinflammatory cytokine expression levels in middle ear tissue of CSOM,COSM+OE-NTLR4,COSM+KD-Nrf2,Control were detected by RT-q PCR method;ELISA method was used to verify the accuracy of the above RT-q PCR results.(7)To verify the efficiency of Nrf2 overexpression and TLR4 knockdown,RT-q PCR was used to detect the difference in the expression level of Nrf2 in the middle ear tissues of AOM,AOM+OE-Nrf2 and TLR4 in the AOM,AOM+KD-TLR4.(8)The difference of proinflammatory cytokine expression levels in middle ear tissue of AOM,AOM+OE-Nrf2,AOM+KD-TLR4,Control were detected by RT-q PCR method;ELISA method was used to verify the accuracy of the above RT-q PCR results.Results: Part I:(1)The expression levels of Nrf2 in the middle ear mucosa tissues of CSOM was significantly higher than that of DOM,Non-OM group,P<0.01,the difference was statistically significant;whereas in DOM,Non-OM group the expression levels of difference was not statistically significant;The expression levels of TLR4 in CSOM middle ear mucosa tissue was significantly lower than that DOM,Non-OM group,P<0.01,the difference was statistically significant;whereas there was no statistically significant difference in the expression levels between DOM and Non-OM groups;The expression levels of TLR2,TLR5 and TLR9 in middle ear tissues of CSOM,DOM,Non-OM were not significantly different.(2)The results of the Western blot method were completely consistent with those of the RT-q PCR method.(3)The expression levels of Nrf2 and TLR4 in the middle ear tissues of CSOM have a statistically significant correlation(Pearson correlation coefficient was 0.608,P<0.0001),TLR4 was negatively correlated by Nrf2.Part II:(1)RT-q PCR results showed that the expression levels of pro-inflammatory cytokines(TNF-α,IL-1β,IFN-γ and IL-6)in middle ear tissue of CSOM were significantly higher than those in DOM,Non-OM group,P<0.01,The difference was statistically significant;whereas there was no statistically significant difference in the expression levels between the DOM and Non-OM groups.(2)The results of the ELISA method are completely consistent with those of the RT-q PCR method.Part III:(1)The expression levels of pro-proliferation related proteins(Cyclin D1 and CDK2)in tissues of CSOM and AOM were significantly lower than those in the Control group,P<0.01,The difference was statistically significant;whereas there was no statistically significant difference in the expression levels between the CSOM and AOM groups.The expression levels of pro-apoptotic proteins(cracked Caspase-3 and Bax)in tissues of CSOM and AOM were significantly higher than those in the Control group,P<0.01,the difference was statistically significant;whereas there was no statistical difference in the expression levels between CSOM and AOM groups.(2)RT-q PCR results showed that the expression levels of pro-inflammatory cytokines(TNF-α,IL-1β,IFN-γ and IL-6)in middle ear tissues of CSOM were significantly higher than AOM,Control group,P<0.01,difference has statistical significane;There was no statistically significant difference in expression levels between the Control and AOM groups.The results of the ELISA method are completely consistent with those of the RT-q PCR method.(3)The expression levels of Nrf2 in middle ear tissue of CSOM was significantly higher than that AOM,Control group,P<0.01,the difference was statistically significant;whereas there was no statistically significant difference in expression levels between AOM and Control groups;The expression levels of TLR4 in middle ear tissue of CSOM was significantly lower than that AOM,Control group,P<0.01,the difference was statistically significant;whereas there was no statistically significant difference in expression levels between AOM and Control groups;The results of the Western blot method are completely consistent with those of the RT-q PCR method.(4)Verification of the transfection efficiency of Nrf2 knockdown and TLR4 overexpression vector.the expression levels of Nrf2 in the middle ear tissue of CSOM+KD-Nrf2 was significantly lower than that CSOM group,P<0.01,the difference was statistically significant;The expression levels of TLR4 in the middle ear tissue of CSOM+OE-TLR4 was significantly higher than that CSOM group,P<0.01,the difference was statistically significant.(5)The expression levels of TLR4 in the middle ear tissues of CSOM+KD-Nrf2 was significantly higher than that CSOM group,P<0.01,the difference was statistically significant;whereas between the Control,CSOM+KD-Nrf2 group,there was no significant difference in the expression levels;The results of the Western blot method are completely consistent with those of the RT-q PCR method.The expression levels of Nrf2 in COSM+OE-TLR4 and CSOM group was not statistically significant,P>0.01;the expression level of Nrf2 in COSM+OE-TLR4 group was significantly higher than that Control group,the difference was statistically significant,P <0.01;The results of the Western blot method are completely consistent with those of the RT-q PCR method.(6)RT-q PCR results showed that the expression levels of pro-inflammatory cytokines in the middle ear tissue of CSOM were significantly higher than CSOM+KD-Nrf2,CSOM+OE-TLR4,Control group,P<0.01,the difference was statistically significant;whereas between the CSOM+KD-Nrf2,CSOM+OE-TLR4 group,there was no significant difference in expression levels.The results of the ELISA method are completely consistent with those of the RT-q PCR method.(7)Verification of the transfection efficiency of Nrf2 overexpression and TLR4 knockdown vector in the middle ear tissue of AOM mice.the expression levels of Nrf2 in the AOM+OE-Nrf2 group was significantly higher than that in the AOM group,P<0.01,difference has statistical significane;The expression levels of TLR4 in the AOM+KD-TLR4 group was significantly lower than that in the AOM group,P<0.01,the difference was statistically significant.(8)RT-q PCR results showed that the expression levels of pro-inflammatory cytokines in the middle ear tissues of the AOM+KD-TLR4 and AOM+OE-Nrf2 groups were significantly higher than that In the AOM,Control group,P<0.01,the difference was statistically significant;whereas in the AOM+KD-TLR4,AOM+OE-Nrf2 group,there was no statistically significant difference in expression levels.The results of the ELISA method are completely consistent with those of the RT-q PCR method.Conclusion:(1)The high expression of Nrf2 in the middle ear tissue of CSOM was related to the pathogenesis of CSOM.The low expression of TLR4 in the middle ear tissue of CSOM was related to the pathogenesis of CSOM.The expression levels of Nrf2 and TLR4 in the middle ear tissue of CSOM was negatively correlated.TLR4 targetingly downregulated by Nrf2 was associated with the pathogenesis of chronic suppurative otitis media.(2)The high expression of pro-inflammatory cytokines(TNF-α,IL-1β,IFN-γ and IL-6)in CSOM was related to the pathogenesis of CSOM,and there was chronic inflammation in CSOM.The Nrf2/TLR4 pathway maintains chronic inflammation in CSOM,but chronic inflammation plays a crucial role in the pathogenesis of CSOM.(3)The high expression of Nrf2 in the middle ear tissue of CSOM was related to the pathogenesis of CSOM.TLR4 was targeted and regulated by Nrf2,the expression levels of TLR4 in CSOM was negatively regulated by Nrf2,but changes in TLR4 levels do not affect Nrf2 expression levels.Nrf2 targeting down-regulation of TLR4 contributes to the pathogenesis of CSOM by maintaining chronic inflammation.In the regulation of CSOM process TLR4 was showed that the high expression of TLR4 eliminated the chronic inflammation of CSOM,whereas knocking down of TLR4 promoted the transition of AOM-CSOM,indicating that the weakening of TLR4 contributed to the pathogenesis of CSOM.Knockdown of Nrf2 can reduce the chronic inflammation of CSOM,and the high expression of Nrf2 promotes the chronic inflammation of AOM,indicating that Nrf2 plays a role in the transition of AOM to CSOM.Collectively,we identified that knock-down of Nrf2 attenuated CSOM progression by reversing chronic inflammation through up-regulating TLR4.This study uncovered the underlying mechanisms of AOM-CSOM transition and may provide potential biological agents for the treatment of clinical CSOM. |
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