Study On The Role And Mechanism Of Clarithromycin Hydrogel In Anti-inflammatory Treatment Of Sinusitis

Clarithromycin(CAM)has both antibacterial and anti-inflammatory effects,and its anti-inflammatory effect in the treatment of sinusitis is its unique advantage,but the molecular mechanism is not clear.The effect of CAM on paranasal sinus mucosa and its epithelial cells(HNEp C)is not further studied.In this study,we developed an injectable and self-healing hydrogel sustained-release clarithromycin CAM-Lips@Hydrogel,to study the antibacterial and anti-inflammatory effects of local application on paranasal sinus mucosa.We studied the relationship between its anti-inflammatory effect and HNEp C's Toll-like receptor 2(TLR2)IL-1 receptor(IL-1R)/NF-kappa B,MAPK signal pathway.It is assumed that its regulation of transforming growth factor kinase 1(TAK1)is a mechanism of antiinflammatory effect.Part I Injectable,self-healing CAM-Lips@Hydrogel in the treatment of ABRS?Objective?The injectable,self-healing and CAM-liposome-loaded PEG hydrogel(CAM-Lips@Hydrogel)was developed to treat acute bacterial sinusitis(ABRS)rabbit model.The anti-bacterial and anti-inflammatory effects of CAM on ABRS paranasal sinus mucosa were tested.?Methods?Based on the high affinity and reversible binding of sulfhydryl groups to silver ions,a 4-arm sulfhydryl PEG hydrogelcapable of injecting,self-healing and carrying CAM(and budesonide,BUD)(CAM-Lips@Hydrogel/CAM+BUD-Lips@Hydrogel)was successfully developed.The ABRS models of New Zealand rabbits were established by S.pneumoniae.The rabbit model of ABRS was treated by maxillary sinus injection of CAMLips@Hydrogel.The anti-bacterial and anti-inflammatory effects of CAM on paranasal sinus mucosa were evaluated by bacterial detection,morphological observation,HE staining.And the IHC,PCR,Western Blot were used to detect the inflammatory indexes of paranasal sinus mucosa,such as IL-1,IL-6,IL-8,TNF-?.?Results? CAM-Lips@Hydrogel has excellent properties such as self-healing,biodegradability,slow drug release,moderate expansion coefficient and changing viscosity with the change of shear force,so it is suitable for injection.When the drug-loaded material was used to treat ABRS rabbit model,it was found that either single load CAM or combined with BUD could effectively inhibit the inflammatory reaction of local paranasal sinus mucosa,so to reduce sinus edema and improve sinus drainage.In addition,it did not produce significant side effects in the whole body.The IL-1,IL-6,IL-8 and TNF-? of paranasal sinus mucosa were significantly down-regulated.?Conclusion?CAM plays both anti-bacterial and anti-inflammatory roles in the local application of ABRS paranasal sinus mucosa.Part II Anti-inflammatory effect of CAM on paranasal sinus mucosa of CRS in vitro?Objective?To examine the difference of anti-inflammatory effect of CAM on paranasal sinus mucosa of CRS patients classified according to Ig E+ and Ig E-pathological types.?Methods?According to the pathological classification of Ig E+ and Ig E-,CRS was divided into two groups.Ethmoid sinus,maxillary sinus and other mucosal tissues were taken during FESS and cultured in vitro,while CAM intervention.HE staining was performed 24 hours later,and m RNA levels of MAPK and NF-?B signal pathway factors were measured by Real-time PCR.?Results?CAM down-regulates several key factors of MAPK and NF-? B signal pathway of CRS Ig E-,especially the common nodes of MAPK and NF-? B signal pathway,ASK-1,TAK1 and TPL-2,but has almost no effect on CRS Ig E+.?Conclusion?The anti-inflammatory effect of CAM in CRS paranasal sinus mucosa is related to the pathological type of Ig E.Part III The anti-inflammatory effect of CAM on HNEp C through TAK1-mediated NF-?B and MAPK signaling pathways.?Objective?To study the effect of CAM on LTA-stimulated HNEp C's inflammatory response,the effect of CAM on NF-?B and MAPK signaling pathway of LTA-stimulated HNEp C,and the effect of CAM on TAK1 activity of HNEp C.?Methods? The intervention of CAM to the HNEp C inflammatory response stimulated by LTA was tested.HNEp C was treated with TAK1 inhibitor N25 and then stimulated with LTA,and the corresponding indexes such as NF-?B p65?phospho-ERK/JNK?phospho-p38 were detected to verify the regulatory effect of TAK1.The specific phosphorylation antibody was used to detect the conserved threonine site of TAK1 Thr178 and Thr184,aimed to verify the effect of CAM on the TAK1 activity of LTA-stimulated HNEp C.?Results?It was confirmed that CAM played an anti-inflammatory role in LTAstimulated HNEp C,with the down-regulated inflammatory factors mainly included IL-6 and IL-8.In the study of inflammatory signal pathway,it was found that CAM down-regulated the DNA binding activity of NF-?B p65 in HNEp C induced by LTA.CAM down-regulated the protein levels of phospho-ERK/JNK and phospho-p38 of LTA-stimulated HNEp C.CAM significantly inhibited the TAK1 phosphorylation of HNEp C by regulating the phosphorylation of Thr184 and Thr178.?Conclusion?CAM can inhibit the inflammation of HNEp C induced by LTA.CAM not only played an anti-inflammatory effect by affecting NF-?B signal pathway of LTAstimulated HNEp C,but also by affecting the MAPK signal pathway.This experiment also verified that CAM acts on the common node of TAK1 to regulate the NF-?B and MAPK signaling pathways of LTA-induced HNEp C inflammation.