Experimental Study On The Mechanism Of Panax Notoginseng Saponins Alleviating Sudden Hearing Loss Through HIF-1α/HDAC2 Signaling Pathway

Background:Sudden Sensorineural Hearing Loss(SSNHL)is short for Sudden deafness.Its etiology is unknown and its pathogenesis is complex.Among them,hair cell damage is one of the main pathogenesis of SSNHL."Huayu Tongqiao" is the main treatment for SSNHL,panax notoginseng is a commonly used blood-activating drug for SSNHL treatment.Panax notoginseng saponins(PNS),the main component of Panax notoginseng,has been reported lately that it can treat SSNHL effectively.Our team constructed the in vivo and in vitro model of sudden hearing loss induced by Lipopolysaccharide(LPS)in the early stage.Studies have found that the expression level of Histone Deacetylase 2(HDAC2)is closely related to the prognosis of SSNHL,and it can regulate the apoptosis of cochlear hair cells induced by Oxidative Stress(OS)by regulating downstream miR-204-5P.As an upstream gene,hypoxia inducible factor 1α(HIF-1α)has a reverse regulation relationship with HDAC2.In addition,our previous study also found that PNS can reduce cisplatin-induced cochlear hair cell damage through the AKT/Nrf2 pathway.Related studies have also shown that PNS is closely related to HIF-1α mediated Reactive Oxygen Species(ROS),and the circRNA--circPPP1CB is highly expressed in cisplatin-induced mouse renal injury model.It is speculated that it may promote the ototoxicity,nephrotoxicity and neurotoxicity induced by exogenous chemicals in mice.In this study,the round window membrane was predigested by collagenase Ⅰ,LPS was injected into the round window niche and then introduced into the inner ear through the round window membrane.SSNHL mouse model was constructed,and the protective effect of PNS on hearing was determined by intraperitoneal injection of PNS in mice.Then,at the cellular level,transfection technology was used to regulate the expression levels of related genes,and the molecular mechanism of LPS induced House Ear Institute-Organ of Corti 1(HEI-OC1)cell damage was explored.In the end,this study verified the mechanism of PNS regulating HIF-lα/HDAC2 pathway through circPPP1CB to reduce LPS-induced apoptosis of HEI-OC1 cells,and provided a theoretical basis for the treatment of SSNHL by panax notoginax "Huayu Tongqiao" method.Part Ⅰ:Effect of PNS on hearing protection of LPS-induced SSNHL in miceObjective:To explore the feasibility of using collagenase I to predigest round window membrane and introduce LPS into round window to construct SSNHL mouse model characterized by inflammatory response,to clarify the protective effect of PNS on LPS-induced hearing loss in mice,and to provide a basis for future related model construction and the introduction of related vectors into the inner ear.Methods:(1)Twenty healthy C57BL/6N mice were randomly divided into LPS group and Artificial Perilymph(AP)control group with 10 mice in each group.All mice were subjected to Auditory Brainstem Response(ABR)test before surgery,then the round window niche was exposed via retroauricular path,1 μl collagenase I predigested the round window membrane,10 minutes later,1 μl LPS solution was injected into the circular window niche,and the round window niche was filled with gelatin sponge.Pour 2 μl LPS solution into gelatin sponge.ABR test was performed on the 3rd postoperative day.(2)After the cochlea was removed,paraffin section was stained with Hematoxylin-eosin(HE)to observe the morphological and structural changes of the cochlea.(3)After cochlea was removed,immunofluorescence staining was performed on basement membrane to observe the damage of hair cells.(4)A total of 40 healthy C57BL/6N mice were randomly divided into four groups with 10 mice in each group:PNS(15.67 mg/kg)10,PNS(31.35 mg/kg)10,PNS(62.7 mg/kg)10,normal saline(NS)group 10,after continuous intraperitoneal injection for seven days,LPS was used to establish the SSNHL mouse model,and ABR thresholds were detected before surgery and on the third day after surgery.Results:(1)The mean ABR Ⅲ wave response threshold of each frequency in AP group was 38.25±2.877 dB SPL before surgery,and the mean ABR threshold of each frequency in the operative ear was 40.00±3.85 dB SPL on the third day after surgery,the difference was not statistically significant(P>0.05,n=10),the ABR thresholds at 4kHz,8kHz,16kHz and 32kHz had no significant change from those before surgery,and the difference was not statistically significant(all P>0.05,n=10).The mean ABR Ⅲ wave response threshold of each frequency in LPS group was 39.25±3.01 dB SPL before surgery,and the mean ABR threshold of each frequency in operative ear was 61.25 ±4.05 dB SPL on the third day after surgery,which was significantly higher than that before surgery,and the difference was statistically significant(P<0.001,n=10),and ABR thresholds at 4kHz,8kHz,16kHz and 32kHz were significantly changed after surgery compared with those before surgery,with statistically significant differences(all P<0.001,n=10).(2)He staining of the cochlea showed significant changes in the morphology and structure of the cochlea in the LPS group,while no significant changes in the structure of the cochlea system were observed in the AP group.(3)Immunofluorescence staining of cochlear basement membrane showed that compared with AP group,hair cells of the LPS group were shed obviously,and the structure of outer hair cells was disordered.(4)PNS has a protective effect on hearing loss induced by LPS in mice,and the protective effect of PNS on hearing is dose-dependent.Conclusions:Collagenase I was used to predigest the round window membrane,LPS was injected into the round window niche and then introduced into the inner ear through the round window membrane,leading to the occurrence of SSNHL in mice.Moreover,this model construction method had no significant damage to the hearing of mice,suggesting that this method can construct the SSNHL mouse model characterized by inflammation.It was demonstrated that PNS can protect LPS-induced hearing loss in mice.Part Ⅱ:The molecular mechanism of apoptosis induced by LPS in HEI-OC1 cellsObjective:To investigate the molecular mechanism of HEI-OC1 apoptosis induced by LPS.Methods:(1)The cultured HEI-OC1 cells were treated with different concentrations of LPS.After 48h,the effects of different concentrations of LPS on cell activity and apoptosis were determined by CCK8,flow cytometry and TUNEL staining.Western blot assay was used to detect the protein expression levels of pro-apoptotic genes Cleaved caspase3,Cleaved caspase9 and Bcl-2 in HEI-OC1 cells treated with different concentrations of LPS.The cultured HEI-OC1 cells were treated with LPS at different time lengths,and the effects of different time gradients on cell activity and apoptosis were determined by CCK8,flow cytometry and TUNEL staining.Western blot assay was used to detect the protein expression levels of pro-apoptotic genes Cleaved caspase3,Cleaved caspase9 and apoptotic inhibitory gene Bcl-2 in HEI-OC1 cells treated with different LPS duration.(2)HEI-OC1 cells were divided into LPS group and normal oxygen control group,and the mRNA and protein expression levels of HDAC2 in the cells were detected by RT-qPCR and Western blot assay.(3)The cells were divided into control group,LPS group,LPS+Negative Control(NC)group and LPS+HDAC2 group by overexpression of HDAC2 transfection.HDAC2 mRNA and protein expression levels in each group were detected by RT-qPCR and Western blot assay.Cell viability of each group at each time point was determined by MTT assay.Cell apoptosis levels in each group were detected by AnnexinV/PI staining and TUNEL staining.The protein expression levels of pro-apoptotic genes cleaved caspase3,Bax and apoptosis inhibitory gene Bcl-2 in each group were detected by Western blot assay.(4)HEI-OC1 cells were divided into siRNA group,si-HDAC2 group and si-HDAC2+miR-204-5p inhibitor group by small interfering RNA transfection technology,and the mRNA expression of miR-204-5p in each group was detected by RT-qPCR.(5)The expression of HDAC2 was up-regulated by plasmid transfection.HEI-OC1 cells were divided into NC group,HDAC2 group and HDAC2+miR-204-5p mimic group.The activity of cells in each group at each time point was detected by MTT assay.(6)By plasmid transfection technology,HEI-OC1 cells were divided into NC group,LPS group,LPS+NC inhibitor group,and LPS+miR-204-5p inhibitor group.Cell viability and apoptosis were determined by MTT assay,AnnexinV/PI staining and TUNEL staining,and the protein expression levels of pro-apoptotic genes cleaved caspase3,Bax and Bcl-2 in each group were detected by Western blot.Results:(1)The treatment of 2.5μg/ml LPS for 12 hours resulted in increased apoptosis and decreased cell activity of HEI-OC1 cells.Moreover,the activity and degree of apoptosis of HEI-OC1 cells were positively correlated with LPS concentration and action time.(2)HDAC2 expression in HEI-OC1 cells in LPS group was significantly lower than that in control group.(3)LPS promoted the apoptosis of HEI-OC1 cells by inhibiting the activity of HDAC2,and overexpression of HDAC2 could significantly improve the survival rate of HEI-OC1 cells and reduce the apoptosis rate of HEI-OC1 cells.(4)HDAC2 was involved in regulating the expression of miR-204-5p in HEI-OC1 cells,and there was an inverse regulation relationship between HDAC2 and miR-204-5p.MiR-204-5p is a key gene in the apoptosis of HEI-OC1 cells induced by LPS.HDAC2 enhances cell activity and reduces cell apoptosis by inhibiting the activity of downstream miR-204-5p.Conclusions:LPS induced apoptosis of HEI-OC1 cells by inhibiting HDAC2 and activating miR-204-5P downstream of HDAC2.Part Ⅲ:Study on the protective mechanism of PNS against LPS-induced apoptosis of HEI-OC1 cellsObjective:To verify the mechanism of PNS in reducing the apoptosis of HEI-OC1 cells induced by LPS.Methods:(1)By adding different drugs,the cells were divided into four groups:LPS-/PNS-,LPS-/PNS+,LPS+/PNS-,and LPS+/PNS+.Cell activity and apoptosis were detected by CCK8,flow cytometry and TUNEL staining.Protein expression levels of pro-apoptotic cleaved caspase3,cleaved caspase9 and Bcl-2 were detected by Western blot.(2)RT-qPCR was used to detect the changes of circPPP1CB mRNA expression in HEI-OC1 cells treated with different LPS/PNS.The effect of circPPP1CB on the apoptosis of HEI-OC1 cells was detected by CCK8,flow cytometry and TUNEL staining.The effect of circPPP1CB on the expression of apoptosis-related proteins in HEI-OC1 cells was detected by Western blot.(3)CircPPP1CB was up-regulated and down-regulated,and HIF-1α protein expression in each group was detected by Western blot.SH-NC,SH-circPPP1Cb-1,SH-HIF-1α-1 and SH-circPPP1Cb-1+SH-HIF-1 α-1,CCK-8,flow cytometry and TUNEL staining were used to detect the changes of cell activity and apoptosis levels in HEI-OC1 cells.The protein expression levels of pro-apoptotic genes cleaved caspase3,cleaved caspase9 and Bcl-2 were detected by Western blot.(4)The expression of HDAC2 in HEI-OC1 cells treated with different LPS/PNS was detected by Western blot.HDAC2 was inhibited,cell activity and apoptosis were detected by CCK8,flow cytometry and TUNEL staining,and the expression of apoptotic related proteins in HEI-OC1 cells with or without HDAC2 inhibition was detected by Western blot.HIF-la was inhibited,and the mRNA and protein expression levels of HDAC2 in cells were detected by RT-qPCR and Western blot assay.The binding of HIF-1α and HDAC2 promoter in HEI-OC1 cells was verified by DNA pull down and ChIP assay.Results:(1)PNS could inhibit LPS-induced HEI-OC1 death and apoptosis.(2)In LPS-induced HEI-OC1 cells,PNS plays a protective role by promoting the expression of circPPP1CB.(3)HIF-1α is regulated by the negative feedback of circPPP1CB,and down-regulation of HIF-1αin cells can partially rescue the cell death caused by the decreased expression of circPPP1CB.(4)In HEI-OC1 cells,HIF-1α inhibited the expression of HADC2,leading to cell death.Conclusions:PNS can reduce the apoptosis of HEI-OC1 cells induced by LPS by activating circPPP1CB to regulate HIF-1α/HDAC2 pathway.