The Protective Effect And Mechanism Of P1N1 In Senescent Hair Cells

Age-related hearing loss(ARHL)or presbycusis is characterized by an age-dependent decline of auditory function.ARHL is usually start with the loss of high-frequency detection.It is the most common cause of hearing loss in the world.Long-term hearing loss not only affects daily communication,but also causes depression and cognitive impairment in the elderly.Thus far,there is no effective treatment available for ARHL.Therefore,it is important to clarify the underlying mechanism of ARHL and explore new methods to inhibit its development.In this research,we used the serum of patients with ARHL,the serum and cochleae of C57BL/6 mice,auditory HEI-OC1 cells as the research objects.To explore the role of PIN1 in senescent hair cells and HEI-OC1 cells,which will provide a basis for further elucidating the pathogenesis of ARHL and its targeted therapy.The experiment is divided into the following three parts:Part One The expression of PIN1 is decreased in the serum of patients with ARHL,in the serum and cochleae of aging C57BL/6 miceObjective:Analyze the expression of PIN1 in the serum of ARHL patients,the serum of aging mice and the cochlea and its relationship with hearing impairment,and preliminarily explore the role and significance of PIN1 in the onset of presbycusis.Methods:1.We collected the serum of patients with presbycusis and healthy control group.The enzyme-linked immunosorbent assay was used to detect the expression level of PIN1,ROS and Klotho protein in human serum.2.We prepared two groups of C57BL/6 mice,two months old and 12months old male mice.6 mice in each group.Serum and cochleae were collected after the brainstem evoked potential(ABR)test.We used enzyme-linked immunosorbent assay(ELISA)to detect the expression level of PIN1,ROS and Klotho protein in mice serum.?-Galactosidase staining method was used to detect the senescence of cochlear hair cells and spiral ganglion cells in each group.Immunohistochemistry and immunofluorescence methods were used to detect the expression of PIN1 in cochlear hair cells and spiral ganglion cells of mice in each group.Results:1.Hearing test of ARHL patients and C57BL/6 miceThe hearing threshold of patients with ARHL is significantly higher,especially at high frequencies.Not only the high-frequency ABR threshold of 12-month-old mice was significantly higher than that of 2-month-old mice,but also low-and medium-frequency is higher than that of 2-month-old mice,which indicates that the auditory function of 12-month-old mice has decreased.2.The levels of PIN1 and Klotho were decreased and the level of ROS was increased in the patients ARHL and old mice serum1)ELISA results showed that compared with the control group,the expression level of ROS in the serum of patients in the ARHL group increased.The young control group and the old control group had lower ROS levels,which was not statistically significant.The expression level of anti-aging protein Klotho is reduced in ARHL patients.The Klotho protein expression level in the serum of the two control groups was higher,and there was no statistical significance between them.The expression level of PIN1 protein decreased among ARHL patients,and it is statistically significant compared with the young control group and the old control group.The expression of ROS in human serum was positively correlated with hearing threshold,while the expression of Klotho and PIN1 was negatively correlated with hearing threshold.2)ELISA results showed that compared with the control group,the serum ROS level of aged C57BL/6 mice increased.The levels of Klotho and PIN1 decreased in the serum of aged C57BL/6 mice.The expression of ROS in mouse serum is positively correlated with the hearing threshold,and the expression of Klotho and PIN1 is negatively correlated with the hearing threshold.3.The number of senescent cells increased in the aged C57BL/6 mice cochleaeThe results of?-galactosidase staining showed that there were more?-galactosidase(SA-?-gal)positive cells in spiral ganglion cells(SGCs)and hair cells(HCs)of old mice than in young mice Mice.4.PIN1 expression was markedly declined in the HCs and SGCs of old mice.IF and IHC analysis showed that the positive expression of PIN1 protein was located in the cytoplasm and nucleus of HCs and SGCs.Compared with young mice,the expression of PIN1 in cochlear hair cells and spiral ganglia of old mice was significantly decreased.Linear analysis showed that the expression of PIN1 in the cochlea was negatively correlated with hearing threshold.Conclusion:The expression of PIN1 and Klotho in serum of patients with ARHL and aged C57BL/6 mice were decreased.The expression of ROS in serum of patients with ARHL and aged C57BL/6 mice was increased.The expression of PIN1 in spiral ganglion cells and hair cells of old mice was decreased.It is suggested that the decrease of PIN1 expression may be related to the occurrence and development of presbycusis.Part Two Decreased PIN1 expression mediates senescence of cochlear hair cells and HEI-OC1 cellsObjective:This part of the study takes adult C57BL/6 mouse serum,cochleae and HEI-OC1 cells as the research objects to explore the role of PIN1 in the senescence of hair cells and HEI-OC1 cells.Methods:1.In order to explore the senescence of HEI-OC1 cells induced by H₂O₂through oxidative stress,and the effect on PIN1 protein:First,the cells were used in different concentrations(0mM,1mM,2mM,3mM,4mM,5mM)H₂O₂ treatment for 2h,MTS method was used to detect cell viability.The IC50 was used to determine that the concentration of H₂O₂ 1mM for 2h stimulated HEI-OC1 cell senescence.Then,immunofluorescence method was used to detect the expression of PIN1 protein,Western blot method was used to detect the expression of PIN1,p16,p21,p53,p-p53 protein,?-galactosidase staining method was used to observe cell senescence.2.To detect the effect and possible mechanism of PIN1 in the senescence of HEI-OC1 cells induced by hydrogen peroxide.The conventionally cultured HEI-OC1 cells were divided into control group,H₂O₂ group,H₂O₂+OE-PIN1group and H₂O₂+NC group.Western blot method was used to detect the expression of PIN1,p16,p21,p53,p-p53 protein,and the cells were collected for?-galactosidase staining and ROS content detection.3.HEI-OC1 cells were briefly exposed to juglone(1,5,10?m for 45min).Western blot was used to detect the expression of PIN1 and senescence-related proteins in the cells.Senescence?-Galactosidase staining detects cell senescence.4.2 months male mice were randomly divided into control group,juglone group,DMSO group.Intraperitoneal injection of juglone(1mg/kg,three times a weekly for 4 weeks)5.We used enzyme-linked immunosorbent assay(ELISA)to detect the expression level of PIN1 protein in mice serum.6.Senescence?-Galactosidase staining method was used to detect the senescence of cochlear hair cells in each group.Results:1.The effect of H₂O₂ on the survival rate of HEI-OC1 cellsCompared with the Control group,after stimulating cells with 1,2,3,4,5mM H₂O₂ for 2h,observe the relative cell survival rate and calculate IC50=1.17mM.2.H₂O₂ can induce senescence and down-regulation of PIN1 expression in HEI-OC1 cells1mMH₂O₂ continued to stimulate HEI-OC1 cells for 2h.Compared with the control group,the percentage of SA-?-Gal positive cells in HEI-OC1 cells treated with H₂O₂ increased significantly.The expression of senescence-related proteins p-p53,p21 and p16 increased,while the expression of PIN1protein decreased significantly.3.PIN1 overexpression can inhibit the senescence of HEI-OC1 cells induced by H₂O₂We transfected HA-PIN1 overexpression plasmid into HEI-OC1 cells.Overexpressed PIN1 caused a significant reduction in the expression of p-p53,p21 and p16.The percentage of SA-?-gal positive cells was also reduced in PIN1 overexpression group.4.The effect of juglone on the expression of PIN1 and related senescent proteins in HEI-OC1 cellsAfter using the PIN1 inhibitor juglone,the expression of PIN1 in cells was significantly decreased,while the expression of senescence-related proteins p-p53,p21 and p16 was significantly increased,and the percentage of SA-?-gal positive cells was also significantly increased.Western blot results showed that there was no difference between the groups when the cells were treated with the same dose of solvent DMSO.5.The effect of juglone on hearing of C57BL/6 mice and the effect of serum PIN1Compared with the control group,the juglone group had an increase in the full-frequency hearing threshold,but mainly high frequency.ELISA analysis showed that compared with the control group,the serum PIN1expression level of mice in the juglone group was significantly reduced.6.Juglone can cause senescence of cochlear hair cells in C57BL/6 miceIn the hair cells of the cochlea of each group of mice,there were more?-galactosidase(SA-?-gal)positive cells in the hair cells(HCs)of juglone-treated mice than in the solvent control group.Conclusion:PIN1 mediates the senescence of cochlear hair cells and HEI-OC1 cells.Overexpression of PIN1 inhibits the senescence of HEI-OC1 cells;inhibiting the expression of PIN1 leads to hearing loss and aggravates the senescence of hair cells and HEI-OC1 cells.Part Three Down-regulation of PIN1 mediates HEI-OC1 cell senescencethrough PI3K/Akt/m TOR signaling pathwayObjective:This part takes HEI-OC1 cells as the research object,uses H₂O₂ to induce senescence of HEI-OC1 cells,overexpresses or inhibits PIN1,inhibits or activates Akt signaling pathway,observes changes in cell senescence,and clarifies that PI3K/Akt/m TOR signaling pathway is in HEI-The role of OC1cells in senescence,to explore whether down-regulation of PIN1 participates in the senescence of HEI-OC1 cells by regulating the PI3K/Akt/m TOR signaling pathway.Methods:1.In order to clarify the possible role of PI3K/Akt/m TOR signaling pathway in the senescence of HEI-OC1 cells,the cells were randomly divided into:Control group,H₂O₂ group,LY294002+H₂O₂ group,SC79+H₂O₂group and H₂O₂+DMSO group.Firstly,the cells were pretreated with LY294002 25?M,SC79 4?g/mL or DMSO for 1h,and then stimulated with 1mMH₂O₂.After h,the cells were collected.Western blot was used to detect the expression of PIN1,p16,p21,p53,p-p53,Akt,p-Akt,m TOR,and p-m TOR,while collecting cells for?-galactosidase staining.2.In order to explore whether PIN1 mediates H₂O₂-induced HEI-OC1cell senescence through the regulation of PI3K/Akt/m TOR signaling pathway,HEI-OC1 cells were divided into Control group,H₂O₂ group,H₂O₂+OE-PIN1group,H₂O₂+NC group and juglone group,the expression of Akt,p-Akt,m TOR,p-m TOR and aging-related proteins were detected by western blot.Results:1.Activation of PI3K/Akt/m TOR signaling pathway is involved in H₂O₂-induced senescence of HEI-OC1 cellsCompared with the control group,the expression of p-Akt and p-m TOR protein in the H₂O₂ group was significantly increased,and the Akt signaling pathway activator SC79 or inhibitor LY294002 was used to pretreat HEI-OC1cells,and then stimulated by H₂O₂.Western blot results sshows that SC79 can significantly up-regulate the expression of p-Akt and p-m TOR protein,while the expression of p-p53,p16,and p21 and the number of SA-?-gal positive cells are significantly increased,while LY294002 can reduce the expression of p-Akt and p-m TOR,p-p53,p16,p21 and the number of SA-?-gal positive cells.Neither Akt activators nor inhibitors affect the expression of PIN1protein in cells.2.PIN1 mediates H₂O₂-induced senescence of HEI-OC1 cells through the regulation of PI3K/Akt/m TOR signaling pathwayCompared with the H₂O₂ group,up-regulating the expression of PIN1protein can reverse the up-regulation of p-Akt and p-m TOR induced by H₂O₂,while the expression of senescent cells and senescence-related proteins decreased.Juglone can reduce the expression of PIN1 protein in HEI-OC1cells,up-regulate the expression of p-Akt and p-m TOR,and increase the expression of senescence and senescence-related proteins in HEI-OC1 cells.Conclusion:PIN1 mediates cell senescence by affecting the PI3K/Akt/m TOR signaling pathway.Overexpression of PIN1 can inhibit the activation of the PI3K/Akt/m TOR signaling pathway,thereby reducing the senescence of HEI-OC1 cells induced by H₂O₂;inhibiting PIN1 activates the PI3K/Akt/m TOR signaling pathway and promotes the senescence of HEI-OC1 cells.Part four ROS down-regulates the expression of PIN1 by mediating p53 phosphorylation,which in turn affects the senescence of hair cells and HEI-OC1 cellsObjective:In this part,adult C57BL/6 mouse serum,cochlea and HEI-OC1 cells are used as the research objects.H₂O₂ is used to induce senescence in vitro.Cells are treated with p53 inhibitor Pifithrin-?(PFT-?)and free radical scavenger NAC to observe PIN1 Expression and expression of senescence and senescence-related proteins,explore the relationship between ROS,p53 and PIN1,and further explore the role of PIN1 in the senescence of hair cells and HEI-OC1 cells.Methods:1.In order to explore the role of p53 in the senescence of HEI-OC1 cells induced by H₂O_2,the conventionally cultured cells were randomly divided into:Control group,H₂O₂ group,Pifithrin-?(inhibitor of phosphorylated p53)+H₂O₂ group and H₂O₂+DMSO group.The cells were pretreated with Pifithrin-?10?M or DMSO for 24 h and then stimulated with 1 m M H₂O₂.After 2 h,the cells were collected.PIN1,p16,p21,p53,p-p53,were detected by Western blot.The cells were collected for?-galactosidase staining and ROS content detection.2.In order to further verify the role of oxidative stress in HEI-OC1 aging and the effect of PIN1 protein expression,the cells were pretreated with ROS inhibitor N-acetyl-L-cysteine(NAC),and then given H2O2 stimulation,DCFH-DA probe method detects the content of ROS in HEI-OC1 cells,Western blotting method detects the expression levels of PIN1 protein and senescence-related proteins,and SA-?-Gal staining detects cell senescence.3.Two-month-old C57BL/6 mice were randomly divided into normal control group,juglone group,juglone+NAC group(n=6),administration method:juglone intraperitoneal injection,(1mg/kg,three times a weekly for 4weeks);oral administration of NAC(1.5g/kg/d,for 4 weeks).The ABR hearing and related indicators were tested separately.Results:1.H₂O₂ down-regulates the expression of PIN1 by mediating p53phosphorylation,thereby affecting senescenceCompared with the H₂O₂ group,when was HEI-OC1 cells were incubated with PFT-?and H₂O₂,the p-p53 protein level decreased,but the PIN1 protein level increased significantly.In addition,PFT-?pretreated cells can reduce the up-regulation of p21 and p16 expression caused by H₂O₂,and the percentage of SA-?-gal positive cells is also reduced.2.H₂O₂ affects the expression and activity of p53 through ROS,which in turn affects senescenceThe ROS level in the H₂O₂ group was significantly higher than that in the control group,but when treated by NAC,the ROS level almost recovered to the control group level,while the expression of p-p53 decreased,the expression level of PIN1 increased,and the expression levels of p21 and p16 decreased.The percentage of SA-?-gal positive cells also decreased.3.NAC treatment can improve hearing loss and cochlear hair cell aging in mice that caused by jugloneCompared with the control group,the juglone group had an increase in the full-frequency hearing threshold,but mainly high frequency.But when the free radical scavenger NAC was given at the same time,the hearing of the mice was significantly improved,and the hearing threshold was decreased.At the same time,the?-galactosidase(SA-?-gal)positive cells in the cochlear hair cells of the NAC treatment group decreased.ELISA results showed that ROS in the serum of mice in the juglone group increased and PIN1 decreased,while the level of serum ROS in the NAC treatment group decreased and the level of PIN1 increased.Conclusion:A large amount of ROS is produced during the senescence of hair cells.Excessive ROS can promote p53 phosphorylation and reduce the expression of PIN1,thereby activating the expression of senescence-related proteins and promoting hair cell senescence.