Role Of Endoplasmic Reticulum Stress In Otitis Media

BackgroundOtitis media(OM)is one of the most common infectious diseases in children,and its incidence is second only to upper respiratory tract infections.There is no accurate report on the incidence of OM in China.According to reports in Europe and the United States,80%of children have at least one episode of OM before the age of 3.Streptococcus pneumoniae is the most common pathogen of OM,and 30%to 60%of OM is caused by Streptococcus pneumoniae.Acute otitis media leading to frequent middle ear infections is still a difficult problem in current clinical treatment.It will not only cause tympanic sclerosis,middle ear adhesions and complicated middle ear cholesteatoma in children,which will lead to progressive decline in children's hearing,and even affect the development of language.Pneumococcal disease(PD)is one of the serious public health problems in the world Otitis media caused by Streptococcus pneumoniae(Spn)is a non-invasive disease that is more harmful to children If not treated or treated improperly,it can induce infantile deafness and affect the development of children's language and other central centers.Clinically,the treatment of otitis media is mainly antibiotics,but Spn can develop resistance to commonly used antibiotics,making clinical treatment more difficult Therefore,establishing a suitable model and exploring the pathogenesis and potential treatment modes of OM have become a top priority for pediatric and ENT clinicians.We urgently need to conduct in-depth research on the pathogenesis of OM and seek therapeutic targets.Endoplasmic reticulum(ER),as a complex membrane network in cells,can not only ensure the homeostasis of cellular calcium and lipids,but also effectively mediate the folding and transport of proteins,especially transmembrane and secretion proteins.It is also the main reservoir of cellular Ca2+.Under certain physiological and pathological conditions(such as hypoxia,oxidative stress,infection,imbalance of Ca2+homeostasis,etc.),the folding of proteins in the endoplasmic reticulum can be inhibited,and unfolded proteins can be aggregated,causing endoplasmic reticulum stress.Endoplasmic reticulum stress can not only induce the expression of glucose-regulated protein GRP78,GRP94 and other endoplasmic reticulum molecular chaperones to produce protective effects,Can also independently induce endogenous cell apoptosis,and ultimately affect the outcome of stressed cells,such as adaptation,injury or apoptosis.The signaling pathways activated by endoplasmic reticulum stress have been extensively studied including the PERK/eIF2 pathway,the IRE1/XBP1 pathway and the three pathways mediated by ATF6.Increasing evidence shows that it is involved in the occurrence of many human diseases and development,such as cerebrovascular disease,cardiovascular disease,neurodegenerative disease,viral hepatitis,diabetes,hearing impairment,etc.,play an important role in the pathogenesis of many human diseases.Studies have found that inflammation triggers endoplasmic reticulum stress,affecting endoplasmic reticulum homeostasis and ultimately leading to inflammatory diseases.Recently,in the research of various inflammatory diseases,some studies have reported the signal cascade between endoplasmic reticulum stress and inflammatory response,especially in inflammatory bowel disease.Moreover,it is well known that overproduction of proteins in the endoplasmic reticulum can trigger inflammation,and inflammation can induce endoplasmic reticulum stress.Previous studies have also shown that inflammatory stimuli can activate UPR and trigger endoplasmic reticulum stress,thereby destroying endoplasmic reticulum homeostasis,increasing inflammatory diseases.In addition to the complexity of the interaction between endoplasmic reticulum stress and inflammation,the response of cells to endoplasmic reticulum stress has components of specific cell types,which determine the adaptation or maladaptation and death of stress conditions.In the maladaptive response to endoplasmic reticulum stress,the inflammatory gene expression program is activated In fact,we have shown that in the process of increasing the intensity of environmental stress,through a new axis of eIF2a kinase PKR interacting with the protein PACT,this non-adaptive overactivation of the pro-inflammatory response occurs.Similarly,it is reported that the activation mechanism of PACT-PKR during endoplasmic reticulum stress is pro-apoptotic.Therefore,recent literature data indicate that endoplasmic reticulum stress can lead to pro-inflammatory conditions or pro-inflammatory conditions can lead to endoplasmic reticulum stress.The role of endoplasmic reticulum stress in the pathogenesis of OM is unclear.Therefore,we tested the existence of endoplasmic reticulum stress and inflammatory gene expression in an experimental OM mouse model,and explored how inhibition of endoplasmic reticulum stress affects the pro-inflammatory response and the pathogenesis of OM.Further study of the relationship between OM and ERS is of great significance to the study of the theory of inflammation and the improvement of cell self-repair mechanisms,and helps to further understand the mechanism of disease occurrence and development,and provides a new theoretical basis for clinical OM disease prevention,and provides OM treatment.Scientific questions to be solved1)How to construct a model that is conducive to observing the long-term effects of otitis media similar to human otitis media,and this model is suitable for exploring the pathological mechanism,inflammatory process and treatment targets of otitis media?2)Does the constructed OM model produce endoplasmic reticulum stress?Does endoplasmic reticulum stress affect OM?3)Can inhibition of endoplasmic reticulum stress reduce inflammation and reduce the occurrence of OM?4)Does endoplasmic reticulum stress play a key or leading role in OM?Research programs and the resultsPart 1:Building a suitable OM model:Characteristics of B6 mouse otitis media induced by peptidoglycan(PGPS)Research protocol:Our previous research proved that inoculation with Galanz-positive bacilli cell wall component peptidoglycan(PGPS can induce middle ear inflammation,thereby creating an OM model that does not require live bacteria or viruses.This model has a high survival rate and is beneficial for long-term observation of effects.PGPS Is a pathogen-associated microbial model(PAMP)that can activate Toll-like receptor 2(TLR2),leading to immune.Research protocol:Our previous research proved that inoculation with Galanz-positive bacilli cell wall component peptidoglycan(PGPS can induce middle ear inflammation,thereby creating an OM model that does not require live bacteria or viruses.This model has a high survival rate and is beneficial for long-term observation of effects.PGPS It is a pathogen-associated microbial model(PAMP)that can activate Toll-like receptor 2(TLR2),leading to the activation of immune response.Intratympanic injection of peptidoglycan(PGPS)(the optimal injection dose of PGPS is 55 ?g/10 ?L,PBS is the solvent)induces C57BL/6J mice to construct otitis media(OM)model,and PBS(10 pL)constructs the control group.Through the ear Speculum observation,auditory brainstem evoked potential(ABR),distortion product otoacoustic emission technology(DPOAE)and tympanometer to evaluate the morphological changes of the tympanic membrane,hearing level and tympanic pressure,and evaluate the characteristics of the middle ear cavity of the otitis media model from morphology and function,And apply RT-PCR HE staining,Western blotting,TUNEL staining and immunohistochemistry to exploring the expression of inflammatory genes and apoptosis-related genes,and to detect the biological characteristics of the PGPS-induced otitis media model.Research results:1.After PGPS induction,it was found that part of the external auditory canal skin and tympanic membrane began to hyperemia on the 1st day,the tympanic membrane was unclear on the 2nd day,the malleus pattern was blurred,and a little purulent secretion was seen in the tympanic cavity on the 3rd day.The inflammation model was established.HE staining found that the middle ear mucosal epithelium of the PGPS group was thickened and the inflammatory cells in the middle ear cavity increased,mainly multinucleated cells.The middle ear cavity of the control PBS group had no inflammatory cell infiltration.2.Auditory function test results suggest that after PGPS induction,the ABR of B6 mice has a statistically significant increase in Click,8Khz,and 16Khz thresholds;the tympanic cavity pressure decreases and becomes a negative pressure state.There was no significant statistical difference between DPOAE and PBS group.3.After PGPS induction,B6 mice inflammation and apoptosis genes cleaved Cas3,TNF-?,IL-6,TLR2 mRNA increased,but IL-1 p had no difference.Western Blot also verified the results of RT-PCR,suggesting that the corresponding inflammation and apoptotic protein expression is also increased,and there is statistical significance.4.Immohistochemistry indicated that the expression of TNF-? in the middle ear mucosal epithelium of the PGPS group was positive.Quantitative analysis indicated that the expression of the PGPS group was significantly increased,and the difference was statistically significant.5.Apoptosis in situ detection TUNEL method to detect the middle ear epithelial cell apoptosis in the middle ear tissue.The number of TUNEL positive middle ear epithelial cells in the PGPS group was significantly more than that in the control group,suggesting that the middle ear epithelial cells of the PGPS group mice Apoptosis.Part 2:The relationship between endoplasmic reticulum stress and otitis mediaResearch protocol:grouping and injection are the same as the first part,using RT-PCR and Western-blot to explore the PGPS induced endoplasmic reticulum related genes ATF6,Cas12,CHOP,BIP mRNA level and related protein expression in the middle ear mucosal epithelium of B6 mice To explore the role of endoplasmic reticulum stress in the occurrence of otitis media,use immunohistochemical methods to detect the expression of the apoptosis-related protein CHOP,explore the endoplasmic reticulum stress-induced cell apoptosis,and participate in the inflammatory mechanism of otitis media;use CellRox kit Detect the generation of ROS in the mucosal epithelium of the middle ear,and explore the relationship between the generation of oxygen free radicals and the occurrence of otitis media.Research results:1.Take the B6 mouse tissues from the PBS group and the PGPS group,and RT-PCR to find the change of ATF6,Gas12,CHOP,and BIP mRNA related to endoplasmic reticulum stress,showing that the expression of B6 mice induced by PGPS is obvious Higher than the PBS group,all have statistical significance(P<0.05).2.Western Blot detection of PGPS-induced B6 mouse middle ear tissues corresponding endoplasmic reticulum stress-related proteins ATF6,Cas12,CHOP,BIP expression also significantly increased,compared with the PBS group,the value was statistically significant(P<0.05).3.Immunohistochemical detection of the apoptotic protein CHOP indicated that the CHOP protein in the B6 mouse group induced by PGPS was expressed in the cytoplasm and nucleus;quantitative analysis showed that the expression of CHOP in the PGPS group was significantly more than that of the control group,and the value was obvious(P<0.05).4.CellRox kit detects the generation of ROS in the epithelial tissue of the middle ear,suggesting that the B6 mouse group has significantly more ROS generation than the control PBS group after PGPS induction,and the fluorescence staining of the middle ear mucosal epithelial cells is positive.Part ?:The protective effect of endoplasmic reticulum stress inhibitor TUDCA on otitis media Research protocol:We selected B6 mice and randomly divided them into three groups:PGPS group(PGPS dose of 55 ?g/10 ?L),TUDCA treatment group(200 ?g TUDCA freshly diluted with 10 ?L PGPS)and PBS group.Through HE staining and ear endoscopy,the middle ear cavity of the three groups of mice was observed;auditory-induced brainstem response(ABR)was used to detect ABR threshold;western-blot was used to detect related inflammation and endoplasmic reticulum stress related protein expressionResearch results:1.ABR examination in TUDCA group was significantly lower than the threshold value of PGPS group in Click,8KHz,16KHz,and the value was statistically significant(P<0.05).Compared with the ABR of the TUDCA group and the PBS group,although the threshold is higher than that of the PBS group,there is no statistically significant difference.2.HE staining showed that the middle ear epithelium of the TUDCA group was compared with the PGPS group,the middle ear mucosal epithelium was thickened and inflammatory cell infiltration decreased,etc.The analysis of the middle ear mucosal epithelium thickness and the ratio of inflammation coverage area indicated that the middle ear of the TUDCA group was compared with the PGPS group Epithelial inflammation was weakened,and the ratio of epithelial thickness and inflammation coverage area decreased,which was statistically significant(P<0.05).3.Western blot indicated that the expression of ATF6,Cas12,CHOP and BIP mRNA of the endoplasmic reticulum stress-related proteins in the TUDCA group was significantly lower than that in the PGPS group.The expression of inflammation-related gene TNF-? in TUDCA group was also significantly reduced.Part ?:To explore the leading role of endoplasmic reticulum stress in OM—the relationship between autophagy,oxidative stress and otitis mediaResearch protocol:Randomly select B6 mice,construct TUDCA,rapamycin(RAP,),PGPS group and PBS mice,use RT=PCR to explore the expression of autophagy genes P62 and LC3 in each group,and explore TUDCA and RAP Inhibition of autophagy mechanism,while detecting the expression of endoplasmic reticulum stress-related genes in each group,exploring the inhibition of RAP on ER-stress;RT-PCR detecting the expression of oxidative stress-related genes in PBS,PGPS group and TUDCA,exploring The role of oxidative stress in PGPS-induced OM;finally clarify the relationship between autophagy,oxidative stress and endoplasmic reticulum stress in the mechanism of otitis media.The results of the study:1.The expression of autophagy-related genes P62 and LC3 in B6 mice induced by PGPS increased,and the autophagy-related genes P62 and LC3 were significantly reduced after treatment with TUDCA and the autophagy inhibitor Rap.Compared with the PGPS group,there are numerical values.Statistical significance(P<0.05);the expression of autophagy genes P62/LC3 decreased more significantly in the TUDCA group and the Rap group,and the difference between the groups was statistically obvious(P<0.05),indicating that TUDCA can inhibit the endoplasmic reticulum response Reduce the occurrence of OM autophagy.2.Consistent with the results of the third part,the TUDCA group compared with the PGPS group,the ERS-related genes ATF6,BIP,and CHOP mRNA were significantly reduced,and the numerical difference between the two groups was statistically significant(P<0.05);The PGPS group endoplasmic reticulum stress-related genes ATF6,BIP,and CHOP mRNA tended to decrease,but the difference between the two groups was not statistically significant(P>0.05);the TUDCA treatment group was compared with the Rap treatment group.BIP mRNA was significantly reduced,and the numerical difference between the groups was obvious(P<0.05),while the reduction of CHOP mRNA was not different between the two groups,indicating that Rap could not inhibit the occurrence of endoplasmic reticulum stress.4.Detection of oxidative stress-related genes.The PGPS group had slightly higher oxidative stress-related protein SOD mRNA expression than the PBS group,but there was no difference in the mRNA expression of Nrf2,GSH-Px,NOD,and Catalase genes;oxidative stress-related after TUDCA treatment The gene is not significantly reduced,suggesting that the oxidative stress mechanism may not be involved in the generation of ROS,indicating that PGPS induced OM oxidative stress did not occur.Innovation and significance1.The successful use of gram-positive bacilli cell wall component peptidoglycan PGPS to induce otitis media model in C57BL/6J mice,and this model is relatively stable,and has a higher survival rate than previous bacteria-induced model mice,which is helpful for observing mice Long-term effects of otitis media.2.Endoplasmic reticulum stress ER-Stress is involved in the occurrence and development of many inflammations.This experiment explains the role and role of ER-Stress in the occurrence and development of otitis media,and explores the mechanism of ER-Stress in otitis media At the same time,inhibition of ER-Stress can effectively reduce middle ear inflammation,indicating that inhibition of ER-Stress can be used as a potential target for the treatment of OM Point,provide a new theoretical basis for the future treatment of otitis media.3.This paper is the first to explore the leading role of endoplasmic reticulum stress in otitis media.Compared with autophagy and oxidative stress,in the PGPS-induced C57BL/6J mouse otitis media model,endoplasmic reticulum stress precedes autophagy.,And can activate the generation of oxygen free radicals under oxidative stress,inhibit endoplasmic reticulum stress and effectively inhibit the occurrence of autophagy,which provides therapeutic prospects for OM treatment.