| Objective: This study mainly observes the clinical efficacy of Acanthopanax tablets in the treatment of sudden deafness of kidney essence deficiency type,which provides a new diagnostic and therapeutic idea for the treatment of sudden deafness by combining traditional Chinese and Western medicine.To observe the protective effect of Eleutheroside E,the main component of acanthopanax,on cisplatin-induced auditory system damage,and to analyze its mechanism of action.Methods: Clinically selected patients with sudden deafness(kidney essence loss type)were treated with Acanthopanax tablets based on basic treatment,and the Chinese medicine symptom scores,pure tone hearing thresholds,Tinnitus Handicap Index(THI)scores,Generalised Anxiety Disorder Scale(GAD-7),Depression Screening Scale(PHQ-9),and Pittsburgh Sleep Quality Index(PSQI)scores of the two groups were compared before and after the treatment,and the safety and the incidence of adverse reactions during the treatment period were observed,to objectively evaluate the clinical efficacy of the patients.In the experimental part,we used network pharmacology to screen the main components of acanthopanax to antagonize cisplatin ototoxicity and protect the damaged auditory system and predicted the main signaling pathways and key targets of its intervention in the damaged auditory system.The mechanism of action of Eleutheroside E was verified by in vivo and in vitro experiments.In the in vivo experiments,a cisplatin-induced C57BL/6J hearing injury model was established,the body weight and dietary changes of mice were observed to understand the general condition of mice,the auditory brainstem evoked potentials and distortion product otoacoustic emission were used to detect the thresholds and amplitude of each frequency,the morphology of the cochlea of mice was observed by H&E staining,and the ultrastructural changes of the mitochondria of the spiral ganglion of mice were observed by transmission electron microscopy so that the protective effect of Eleutheroside E on the impaired auditory system of mice was demonstrated.The protective effect of Eleutheroside E on the damaged auditory system of mice was clarified.4D-Fast DIA quantitative proteomics was performed on mouse cochlear tissues to clarify the key proteins and signaling pathways involved in the effect of Eleutheroside E.In vitro experiments,cisplatin-induced proteomics was established in the cochlea.In vitro experiments,we established cisplatin-induced HEI-OC1 cells and isolated cochlear basement membrane injury models in mice,observed the morphological changes of hair cells and spiral ganglia by immunofluorescence staining,measured the cell survival rate by CCK-8 cell viability assay,and determined the apoptosis by flow cytometry,to clarify the protective effect of Eleutheroside E.We also determined the protective effect of Eleutheroside E by using real-time fluorescent quantitative PCR,protein immunoblotting,enzyme-linked immunosorbent assay,and quantitative proteomic analysis by using4D-Fast DIA.Quantitative real-time polymerase chain reaction,protein immunoblotting,and enzyme-linked immunosorbent assay were used to verify the expression of the MAPK/NF-κB/NLRP3 signaling pathway and the proteins and genes related to pyroptosis.Results: The clinical study showed that both the treatment group and the control group improved the symptoms of the patients,and the total effective rate of the treatment group was 45.71%,and the total effective rate of the control group was 42.86%(P>0.05),the difference was not statistically significant.After the treatment,the tinnitus disability assessment scale(THI)score,generalized anxiety disorder scale(GAD-7),depression screening scale(PHQ-9),Pittsburgh sleep quality index(PSQI)score,and Chinese medicine syndrome score of the patients in both groups improved compared with those before the treatment,and in the tinnitus disability assessment scale(THI)score,generalized anxiety disorder scale(GAD-7),depression screening scale(PHQ-9),Pittsburgh sleep quality index(PSQI)score,and Chinese medicine syndrome score,the patients in the control group showed improvement in their symptoms.PHQ-9),and Pittsburgh Sleep Quality Index(PSQI)improvements were better in the treatment group(p < 0.05).The results of the network pharmacological study indicated that Eleutheroside E is the main component of acanthopanax to antagonize the ototoxicity of cisplatin and that Eleutheroside E may play a protective role against the damaged auditory system through the MAPK signaling pathway and inflammation-related signaling pathway.The results of in vivo studies showed that Eleutheroside E could improve the general condition of mice,and the results of brainstem evoked potential test showed that Eleutheroside E had a significant protective effect against cisplatin-induced hearing loss at 8000,16000,and 32000 Hz,and the results of otoacoustic emission showed that Eleutheroside E could protect the outer hair cells from damage by cisplatin.HE staining results showed that the pre-administration of Eleutheroside E could reduce the damage to the spiral ganglion,vascular stripe,and Corti apparatus.Transmission electron microscopy results showed that the damage of spiral neuron cells and mitochondria of nerve fibers in the treatment group was smaller than that in the model group,and the analysis of proteomics results showed that the MAPK signaling pathway played a key role in the protective process.In vitro experiments,CCK-8 cell viability assay,flow cytometry,and immunofluorescence showed that Eleutheroside E could inhibit the death of HEI-OC1 cells,auditory hair cells,and spiral ganglion neuron cells.The results of WB,q RT-PCR,and ELISA experiments indicated that Eleutheroside E inhibited the activation of MAPK/NF-κB/NLRP3 signaling pathway and reduced the secretion of pro-inflammatory mediators,thus inhibiting the pyroptosis.Conclusion: The results of clinical application showed that the combined use of Acanthopanax tablets had a significant improvement effect on the accompanying symptoms such as sleep disorders,emotional abnormalities,tinnitus,ear stuffiness,and distension,etc.,without decreasing the efficacy of patients with sudden deafness with renal essence deficiency.The experimental results showed that Eleutheroside E could inhibit cellular death by down-regulating the activity of MAPK/NF-κB/NLRP3 signalling pathway,lowering the expression level of pyroptosis-related proteins and the level of inflammatory factors,and thus exerting a protective effect on the damaged auditory system. |
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