The Effect And Mechanism Of MtDNA4834bp Deletion In Model Of Inner Ear Mimetic Aging

Part one: Establishing model of mimetic aging of inner ear with mtDNA mutation in ratObject To explore the establishment of the mimetic aging effect in the inner ear of the rat. Methods Twenty-four wistar rats were randomly divided into two groups: group A (D-galactose group, n=14), were treated with hypodermic 5% D-galactose (150mg.kg-1.d-1) for 8 weeks and then with intraperitoneal saline for 10days; group B (control group, n=10) were given saline only. Auditory brainstem response(ABR) was used to detect the hearing threshold of rats, and colorimetry was used to analyze the activity of the glutathione peroxidase(GSH-PX). The inner ear tissue was harvested and mitochondrial DNA was amplified to identify the 4834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. Results The incidence of mitochondrial DNA 4834bp deletion of group A was100 %(28/28) , while group B was 0. The activity of the GSH-PX in group A(59.07±2.32U) is lower than in group B(142.10±2.21U), the difference between group A and group B is significant(t-test,p=0.000<0.01). 5.36±3.08 dB peSPL variation in ABR threshold was observed in group A, and 6.50±3.37 dB peSPL in group B. The difference in shift of ABR threshold between group A and group B is not significant (t-test,p=0.508>0.05). Conclusion The mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and these rats of model present high incidence of mtDNA4834 deletion. But no hearing loss was found in this model. Part two: The comparison of three ways for extracting nucleic acid from rat's hairObjective To discuss the method of extracting nucleic acid from rat's hair. Methods The method with PCR buffer,SDS-proteinase K or Chelex-100 were used to extract the nucleic acid from rat's hair separately, and the isolated nucleic acid was analyzed by PCR and electrophoresis. Results The success rate of extracting mtDNA from the hair papilla, we found the method with SDS-proteinase K is lower than the other methods (χ1=42.421,χ2=28.800,P<0.001). This is same as the result of extracting nuclear DNA (χ1=49.091,χ2=30.767,P<0.001). While no difference has been found between the method with PCR buffer and with chelex-100(mtDNA:χ=0.296;nuclear DNA:χ=0.048,P >0.05).The method with PCR buffer can't extract nucleic acid from hair shafts, the method with SDS-proteinase K can't extract mtDNA from one or two hairs, the method with Chelex-100 can extract mtDNA and nuclear DNA from single rat's hair or hair shafts. Conclusion The method with Chelex-100 is suitable for extracting nucleic acid from hair.Part three:The incidence of mitochondrial DNA 4977bp deletion mutation among the different tissuesObject To explore incidence of mtDNA4834bp deletion in the inner ear,kidney,brain,blood,skeletal muscle,spleen,heart,liver,lung and hair of model rat and elucidate its potential pathological significance.Meanwhile, to determine possibility of hair instead of blood preparation used in clinical examination of mtDNA deletion. Methods Twenty-four Wistar rats were randomly divided into two groups: group A (D-galactose group , n = 14) ,which were treated with hypodermic 5% D-galactose (150 mg·kg - 1·d - 1 ) for 8 weeks and then with intraperitoneal saline for 10 days,and group B (control group , n = 10) , which were given saline only. The various tissues were harvested and mitochondrial DNA was amplified to identify the 4834bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. Results CD can be examined in various tissue of group A.incidence of CD in inner ear,temporal muscle and liver are highest(100%),kidney and brain are second(92.86%),spleen is 85.71%,heart,lung and hair are 71.43%,blood is the lowest(35.71%).The difference of incidence of CD between blood and inner ear was significant(p=0.001<0.01,two-tailed test,Fisher's test),but it is not significant between other tissue and inner ear(pa=1.000,pb=0.481, pc=0.098,pall>0.05,two-tailed test,Fisher's test)。in group B, CD can be investigated only in liver(20%),brain and kidney(10%),the other tissue was negative. Conclusion Result indicates that D-galactose can induce mtDNA4834bp deletion not only in inner ear, but also in many other tissue.The difference of incidence of CD may concern with degree of caryocinesia and activity of oxidative phosphorylation. Meanwhile, the result indicated that incidence of CD in hair can represent in inner ear more suitably.Hair can be used in clinical examination of mtDNA deletion instead of blood.Part four: The sensitivity to the ototoxicity of aminoglycoside antibiotic of the animal model with mimetic aging in the inner earObject To research the animal model with mimetic aging effect in the inner ear predispose to the ototoxicity of aminoglycoside antibiotic. Methods fifty wistar rats were randomly divided into four groups: group A (D-galactose group, n=14),were treated with hypodermic 5% D-galactose (150mg.kg-1.d-1) for 8 weeks and then with intraperitoneal saline for 10days; group B (D-galactose and kanamycin group, n=14),were given the same dose of D-galactose but kanamycin (500mg.kg-1.d-1.) instead of saline; group C (kanamycin group, n=12) were treated with saline for 8 weeks and then with intraperitoneal kanamycin for 10 days;group D (control group, n=10) were given saline only. Auditory brainstem response(ABR) was used to detecte the hearing threshold of rats, and colorimetry was used to analyze the activity of the GSH-PX. The inner ear tissue was harvested and mitochondrial DNA was amplified to identify the 4834 bp deletion mutation by nested primer polymerase chain reaction (nested PCR) technique. Results The incidence of mitochondrial DNA4834bp deletion mutation of group A was 100 % (28/ 28), group B was 92.86%(26/ 28),and group C or group D was 0. The activity of GSH-PX in group A was 59.07±8.70U,in group B was 63.29±12.40U,in group C was 136.67±9.53U,in group D was 142.10±7.02U. The difference between group A and D is significant (pAD=0.000).while the difference between group A and B is not significant (pAB=0.307), similarly as between group C and group D(PCD=0.151). 5.36±3.08 dB peSPL variation in ABR threshold was observed in group A, 61.79±11.20 dB peSPL in group B, 34.17±4.69 dB peSPL in group C,and 6.50±3.37 dB peSPL in group D.No difference was found between group A and D(pAD=0.398), while the difference in shift of ABR threshold between group B and group C(or group D) is significant(pBD=0.000, pCD=0.000). Conclusion The mimetic aging effect in the inner ear of the rat can be induced by D-galactose, and these rat model present high incidence of mtDNA4834 deletion. The mutation can greatly enhance the sensitivity of the inner ear to the aminoglycoside antibiotic. Part five:The role of nuclear factor in inner ear of rat which sensitivity to the aminoglycoside antibioticObject To investigate mitochondrial copy number in membranous labyrinth of inner ear of aminoglycoside antibiotics-induced hearing loss model of rat,the change of mRNA level of nuclear gene encoding NRF-1,PGC-1,mtDNA encoding cytochrome C oxidase COXIII, and the change of protein level of COXIII and mtDNA4834bp deletion and explore interaction between nuclear factor and mtDNA4834bp deletion contribute to sensitivity of the inner ear of rat to aminoglycoside antibiotics. Methods Sixteen Wistar rats were randomly divided into two groups: group A(D-galactose and kanamycin group,n=10) were treated with hypodermic D-galactose (150 mg/kg/d)for 8 weeks and then with intraperitoneal kanamycin (500 mg/kg/d) for 10 days.group B(control group, n = 6) was administrated with saline instead of D-galactose. Auditory brainstem response (ABR) was used to detect the hearing threshold of rats,polymerase chain reaction(PCR) was used to detect inner ear mitochondrial copy number,nest PCR was used to detect the incidence of inner ear mtDNA4834bp deletion,semiquantitative RT-PCR was used to detect the mRNA level of NRF-1,PGC-1 and COXIII and western-blot was used to detect protein level of COXIII in inner ear. Results The incidence of mitochondrial DNA 4834 bp deletion of group A was 100%,while the deletion of group B was 0 . 66.79±7.75 dB peSPL increased in ABR threshold was observed in group A, and 8.33±6.83 dB peSPL in group B. The CN level of group A is 2.75±1.38, group B is 5.76±1.04. The difference between A and B was significant(p<0.01). The mRNA level of NRF-1 and PGC-1 decreased(0.51±0.10 vs 0.94±0.06,2.93±0.98 vs 7.92±1.53, P<0.01). The mRNA level of COXIII didn't change(2.83±0.65 vs 3.15±0.53,P>0.05 ), but protein level decreased(P<0.01). Conclusion There is mutual regulation between nuclear factor and mtDNA, their cooperation effect may be related with sensitivity of the inner ear of rat to aminoglycoside antibiotics.