| PrefaceSecretory otitis media (SOM) is the most common agent that causes acouesthesia decrease, especially on children. SOM raises the risk of otitis media's acutely episode. The SOMs happen in infancy would directly influence the obtaining of language information during the language development process, and would influence the patients'quality of lives even possibly result in lifelong influence to the sick children. It can also cause middle ear accretion, atrophia and sclerosis of tympanic membrane, necrosis of auricular bone, tympanosclerosis, cholesterol granuloma, cholesteatoma and other kinds of complications. The pathogenesy of SOM is still unclear and the reasons of morbility, course and turnover may be multiple factored and syntrophic. Recent researches have discovered that LPS plays an important role in the morbility and protraction of the SOM. The receptor of LPS is Toll like receptor 4 (TLR4), TLR4 receives the stimulus of LPS and activates NF-κB to switch on the target gene expression by the MyD88 dependent pathway then mediates the inflammatory response. The purpose of this study is to observe the expression of TLR4 and its correlation factors in normal rat's and SOM rat's middle ear mucosa through in vivo and vitro experiment, so to justify whether the TLR4 signal passageway participate the morbility of SOM. Previous research found that the remodeling of tracheal tissue, especially the extracellular matrix remodeling, resulted in dysfunction of ventilation on chronic inflammation was the main cause of pulmonary functional lesion. MMPs/TIM and TGF-βare closely correlated with extracellular matrix remodeling. This study was designed to observe the pathological change of the middle ear mucosae especially the extracellular collagen fibers and the expression of MMPs/TIMP, TGF-βin chronic SOM, and to observe whether there was extracellular matrix remodeling in chronic SOM.Materials and method:1. 27 rats were selected and randomly divided into 3 groups. groupⅠ: infused normal sodium into the acoustic vesicles; groupⅡ: infused 1mg/ml LPS 30μl into the acoustic vesicles, group;Ⅲ: infused 1mg/ml LPS 30μl to the acoustic vesicles after the infusing of hemaleucin sealant. On the 3rd day after the operation observed the tympanic membrance with electronic auriscope every 2 days. On the 7th and the 14th preoperative day detected ABR one time respectively. On the 3rd, 7th, 14th, postoperative day took and executed 3 rats after anesthesia respectively, then counted the acoustic vesicles with MEE in auris media meanwhile HE staining to observe morphological change of the acoustic vesicles.2. Selected 36 rats, infused hemaleucin sealant and LPS to the auri laevae while the auris dextra was the blank group. Executed the rats (6 rats each time) on the 1st day, 3rd day, 5th day, 7th day, 12th day and 14th day, got the respectively middle ear mucosae to detecte the expressions of TLR4, inducible nitric oxide synthase (iNOS), IL-8mRNA through RT-PCR and detected the activation of the NF-κB in auris media mucosae by the method of immunohistochemistry.3. Cultured the rat middle ear mucosae endothelial cells in vitro. Divided the culture dishes in which were the endothelial cells culturing into 6 groups: blank group, PDTC group: added the PDTC and the concentration was 0.05mM, Erythromycin group: added the Erythromycin and the concentration was 0.1mM, LPS group: added the LPS and make sure the LPS concentration was 100ng/ml in the culture solution, LPS+PDTC group: added the LPS and PDTC, the concentration was 0.05mM. LPS+Erythromycin group: added the LPS and Erythromycin, the concentration was 0.1mM. Detected the expression of TLR4, iNOS, IL-8mRNA in the endothelial cells the 4th hour after the administration through RT-PCR. Use immumofluorescence method to detect the expression of NF-κB65, TGF-β1 on the 1st, the 4th hour and ELISA to detect the concentration of TNF-αin the culture solution on the 1st, 4th, 24th hour.4. Selected 24 rats, infused hemaleucin sealant and LPS to the auri laevae as SOM group while infused the normal saline to auris dextra as blank group. Executed the rats the 1st, 7th, 20th day, obtained the acoustic vesicles then fixed, decalcificated and prepared paraffin sections. Masson staining determined the percentage areas of the stained collogen constituent in middle ear mucosae, detected the expression of MMP2, MMP9, TIMP1, TGF-β1in middle ear mucosae by the method of immunohistochemistry.Result1. The morphological changes of tympanic membrane were observed in rats in groupⅡand groupⅢafter the operation and there were more rats with morphological changes in groupⅢthan in groupⅡ. ABR test results showed that the audibility threshold was obviously increased in both groupⅡand groupⅢand it was markedly higher in groupⅢcompared with groupⅡ. The number of the acoustic vesicles with MEE was larger in groupⅢthan that in groupⅡ. Inflammatory reaction on the 7th and 14th postoperative day in groupⅢwas more serious than groupⅡ, as showed in HE Staining.2. RT-PCR detection displayed that the expression of TLR4mRNA in middle ear mucosae of normal rats was slight while it was obviously raised in SOM and the expressions of iNOS, IL-8mRNA were similar to TLR4. Immunohistochemistry method showed that the activation of NF-κB in middle ear mucosae was obviously increased in SOM.3. RT-PCR results showed there was no significant deviation on the slight expression of TLR4mRNA in the middle ear mucosae in control group, PDTC group and Erythromycin group, the expression quantity was obviously increased in LPS group, the expression quantities in LPS+PDTC group and LPS +Erythromycin group were higher than normal group while lower than LPS group. The expressions of iNOS and IL-8mRNA were similar to TLR4. There was no significant deviation on NF-κBp65 nucelus aversion percentage in control group, PDTC group and Erythromycin group on the 1st hour by immunofluorescence, it was obviously higher in LPS group compared with control group and there was no significant deviation between LPS+Erythromycin group and LPS group while it was obviously lower in LPS+PDTC group than LPS group. The only difference showed between the 4th hour and the 1st hour was that the nucelus aversion percentage was striking lower in LPS+Erythromycin group compared with LPS group, the rest were identical with the 1st hour. At the 1st hour and the 24th hour there was no significant deviation on the fluorescence intensity of TGF-β1 between control group and PDTC group and in the LPS group and Erythromycin group it was higher than control group, LPS+Erythromycin group was obvious higher than LPS group while it was significant lower in LPS+PDTC group than LPS group (p<0.05). Results in the ELISA showed that the concentration of TNF-αwas lower in LPS+PDTC group than LPS group at each set time and in LPS +Erythromycin group there was no significant deviation with the LPS group on the 1st hour while it was obvious lower on the 4th and the 24th hour.4. Masson Staining showed that the extracellular collogen elements endlessly increased as the lasting of the course of disease, immunohistochemistry showed that the impressions of MMP2, MMP9, TIMP1, TGF-β1 in middle ear mucosae were obviously raised and the constituent of the extracellular collagen fibers was also raised.Conclusion1. It would obtain higher achievement ratio and more protracted course of SOM by using the method that infuse the hemaleucin sealant first then LPS second into acoustic vesicles compared with infusing LPS simply during the manufactures of rat SOM models.2. TLR4 exists in rat middle ear mucosae and the expression of TLR4 obviously rises in SOM and it activates the NF-κB, promotes the expression of downstream IL-8 then initiates SOM. TLR4 exists in mucosal endothelial cells and the TLR4 signal conduction pathway is activated when stimulated by LPS then the inflammatory cytokine generates. The endothelial cells in middle ear mucosae could participate to the inflammatory reactions on multiple identities.3. Both Erythromycin and PDTC are able to inhibit the activation of NF-κB so to start the anti-inflammatory action. PDTC inhibit the activation of NF-κB directly while the Erythromycin may indirectly inhibit the activation of NF-κB by means of increasing TGF-β1expression.4. The phenomenon of extracellular matrix remodeling in middle ear mucosae appears in chronic SOM samples, and it takes place relating to the disequilibrium of TGF-β1and MMPs.
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