葛根素对顺铂致毒小鼠耳蜗钙蛋白酶2表达的影响
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摘要
目的研究葛根素对顺铂致毒小鼠耳蜗钙蛋白酶2(Calpain 2)表达的影响。方法健康BALB/c小鼠60只,随机分成对照组(腹腔注射等量生理盐水)、顺铂组(腹腔注射顺铂4.5mg·kg~(-1)·d~(-1))、顺铂+葛根素组(一侧腹腔注射顺铂4.5mg·kg~(-1)·d~(-1),对侧腹腔注射葛根素200mg·kg~(-1)·d~(-1))和葛根素组(腹腔注射葛根素200mg·kg~(-1)·d~(-1)),每组15只。各组小鼠给药前后进行听性脑干反应(auditory brainstem response,ABR)测试,于停药并完成ABR测试后取动物听泡显微镜下观察耳蜗外毛细胞缺失情况,应用免疫荧光染色和蛋白质印迹技术检测各组耳蜗Calpain 2的表达。结果顺铂组小鼠ABR阈移和外毛细胞缺失率较对照组明显增大(P<0.01),耳蜗内Calpain 2表达较对照组明显增强(P<0.01);顺铂+葛根素组小鼠ABR阈移和外毛细胞缺失率(底回、中回)较顺铂组明显减少(P<0.01),耳蜗Calpain 2表达较顺铂组明显减弱(P<0.01);而葛根素组ABR阈移、外毛细胞缺失率及Calpain 2表达与对照组间差异均无统计学意义(P>0.05)。结论葛根素可有效抑制顺铂所致小鼠耳蜗Calpain 2的高表达,以减轻顺铂的耳毒性,改善小鼠听功能。
Objective To investigate the effects of puerarin on the expression of Calpain 2 in cisplatin-induced mouse cochlea.Methods Healthy BALB/c mice were randomly divided into control group(intraperitoneal injection of saline),cisplatin group(intraperitoneal injection of cisplatin 4.5 mg·kg~(-1)·d~(-1)),cisplatin+puerarin group(intraperitoneal injection of cisplatin 4.5mg·kg~(-1)·d~(-1) and puerarin 200mg·kg~(-1)·d~(-1)),and puerarin group(puerarin 200mg·kg~(-1)·d~(-1) intraperitoneally),with 15 mice in each group.Auditory brainstem response(ABR)was recorded before and after the treatment in each group,and the loss of out hair cell was observed at the same time.The expression of Calpain 2 was detected by immunofluorescence staining and western blot.Results The ABR threshold shift and loss of outer hair cells in the cisplatin group were significantly higher than those in the control group(P<0.01),and the expression of Calpain 2 in the cochlea was significantly higher than that in the control group(P<0.01).The ABR threshold shift and loss of outer hair cells(the basal turn,the middle turn)in cisplatin+puerarin group were significantly lower than those in cisplatin group(P<0.01),and the expression of Calpain 2 in cochlea was significantly lower than that in cisplatin group(P<0.01).Conclusion Puerarin could effectively inhibit the high expression of Calpain 2 in mouse cochlea caused by cisplatin and attenuated cisplatin ototoxicity and improved auditory function.
Objective To investigate the effects of puerarin on the expression of Calpain 2 in cisplatin-induced mouse cochlea.Methods Healthy BALB/c mice were randomly divided into control group(intraperitoneal injection of saline),cisplatin group(intraperitoneal injection of cisplatin 4.5 mg·kg~(-1)·d~(-1)),cisplatin+puerarin group(intraperitoneal injection of cisplatin 4.5mg·kg~(-1)·d~(-1) and puerarin 200mg·kg~(-1)·d~(-1)),and puerarin group(puerarin 200mg·kg~(-1)·d~(-1) intraperitoneally),with 15 mice in each group.Auditory brainstem response(ABR)was recorded before and after the treatment in each group,and the loss of out hair cell was observed at the same time.The expression of Calpain 2 was detected by immunofluorescence staining and western blot.Results The ABR threshold shift and loss of outer hair cells in the cisplatin group were significantly higher than those in the control group(P<0.01),and the expression of Calpain 2 in the cochlea was significantly higher than that in the control group(P<0.01).The ABR threshold shift and loss of outer hair cells(the basal turn,the middle turn)in cisplatin+puerarin group were significantly lower than those in cisplatin group(P<0.01),and the expression of Calpain 2 in cochlea was significantly lower than that in cisplatin group(P<0.01).Conclusion Puerarin could effectively inhibit the high expression of Calpain 2 in mouse cochlea caused by cisplatin and attenuated cisplatin ototoxicity and improved auditory function.
引文
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2张立伟,刘芳芳,于利,等.顺铂对离体培养小鼠耳蜗毛细胞钙蛋白酶表达的影响[J].听力学及言语疾病杂志,2016,24:167.
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4刘芳芳,王爱梅,许涛,等.顺铂对离体培养小鼠耳蜗毛细胞的损伤及凋亡作用[J].南昌大学学报(医学版),2010,50:4.
5 Zhou YX,Zhang H,Peng C.Puerarin:a review of pharmacological effects[J].Phytother Res,2014,28:961.
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9许涛,马婷婷,吴喜迪,等.蛋白磷酸酶1在D-半乳糖诱导老化小鼠耳蜗中的表达[J].听力学及言语疾病杂志,2017,25:176.
10 Yamaguchi T,Yoneyama M,Ogita K.Calpain inhibitor alleviates permanent hearing loss induced by intense noise by preventing disruption of gap junction-mediated intercellular communication in the cochlear spiral ligament[J].Eur J Pharmacol,2017,803:187.
11 Yamaguchi T,Yoneyama M,Hinoi E,et al.Involvement of calpain in 4-hydroxynonenal-induced disruption of gap junction-mediated intercellular communication among fibrocytes in primary cultures derived from the cochlear spiral ligament[J].J Pharmacol Sci,2015,129:127.
12 Ding ZJ,Chen X,Tang XX,et al.Apoptosis-inducing factor and calpain upregulation in glutamate-induced injury of rat spiral ganglion neurons[J].Mol Med Rep,2015,12:1685.
13 Yu L,Tang H,Hua JX,et al.Involvement of calpain-I and microRNA34in kanamycin-induced apoptosis of inner ear cells[J].Cell Biol Int,2010,34:1219.
14黄志辉,黄逸慧,陈慧英,等.大鼠下丘神经元钙蛋白酶1表达与水杨酸钠所致听力损害的关系[J].山东医药,2017,57:29.
15王昌,汤旭磊,陈克明,等.葛根素对过氧化氢致成骨细胞氧化损伤的影响[J].中国骨质疏松杂志,2017,23:1442.
16 Cheng Y,Leng W,Zhang J.Protective effect of puerarin against oxidative stress injury of neural cells and related mechanisms[J].Med Sci Monit,2016,22:1244.
17都君君,刘洪伟.葛根素对妊娠期糖尿病模型大鼠脑组织氧化应激和细胞凋亡的影响[J].中医学报,2018,33:1407.
18孟香红,蒋建平,陈洁,等.葛根素通过抑制Calpain对抗高糖诱导的血管内皮细胞凋亡[J].中国病理生理杂志,2010,26:1992.
19杨巍,刘亚峰,周志强.葛根素对突发性耳聋患者LPO、SOD、6-Keto-PGF-1a)、TXB-2水平的影响[J].中华保健医学杂志,2018,20:144.
20王政林,蔡克万,刘文婷,等.葛根素联合神经节苷脂钠对突发性耳聋患者血液流变学及血管内皮功能的影响[J].中国药业,2018,27:60.
21刘卫红,姚琦,王陈荣,等.葛根素注射液耳后注射联合声频共振治疗老年性聋疗效观察[J].新中医,2013,45:110.
2张立伟,刘芳芳,于利,等.顺铂对离体培养小鼠耳蜗毛细胞钙蛋白酶表达的影响[J].听力学及言语疾病杂志,2016,24:167.
3 Chang L,Wang AM.Calpain mediated cisplatin-induced ototoxicity in mice[J].Neural Regen Res,2013,8:1995.
4刘芳芳,王爱梅,许涛,等.顺铂对离体培养小鼠耳蜗毛细胞的损伤及凋亡作用[J].南昌大学学报(医学版),2010,50:4.
5 Zhou YX,Zhang H,Peng C.Puerarin:a review of pharmacological effects[J].Phytother Res,2014,28:961.
6乔占清.葛根素治疗突发性耳聋的疗效观察[J].中国实用神经疾病杂志,2015,18:104.
7宝东艳,胡真真,李芳,等.葛根素对卡那霉素耳中毒小鼠耳蜗核因子-κB p50活性的影响[J].中国老年学杂志,2016,36:2574.
8宝东艳,王爱梅,卢伟,等.葛根素对卡那霉素耳中毒小鼠耳蜗NF-κB p65活性的影响[J].广东医学,2015,36:1490.
9许涛,马婷婷,吴喜迪,等.蛋白磷酸酶1在D-半乳糖诱导老化小鼠耳蜗中的表达[J].听力学及言语疾病杂志,2017,25:176.
10 Yamaguchi T,Yoneyama M,Ogita K.Calpain inhibitor alleviates permanent hearing loss induced by intense noise by preventing disruption of gap junction-mediated intercellular communication in the cochlear spiral ligament[J].Eur J Pharmacol,2017,803:187.
11 Yamaguchi T,Yoneyama M,Hinoi E,et al.Involvement of calpain in 4-hydroxynonenal-induced disruption of gap junction-mediated intercellular communication among fibrocytes in primary cultures derived from the cochlear spiral ligament[J].J Pharmacol Sci,2015,129:127.
12 Ding ZJ,Chen X,Tang XX,et al.Apoptosis-inducing factor and calpain upregulation in glutamate-induced injury of rat spiral ganglion neurons[J].Mol Med Rep,2015,12:1685.
13 Yu L,Tang H,Hua JX,et al.Involvement of calpain-I and microRNA34in kanamycin-induced apoptosis of inner ear cells[J].Cell Biol Int,2010,34:1219.
14黄志辉,黄逸慧,陈慧英,等.大鼠下丘神经元钙蛋白酶1表达与水杨酸钠所致听力损害的关系[J].山东医药,2017,57:29.
15王昌,汤旭磊,陈克明,等.葛根素对过氧化氢致成骨细胞氧化损伤的影响[J].中国骨质疏松杂志,2017,23:1442.
16 Cheng Y,Leng W,Zhang J.Protective effect of puerarin against oxidative stress injury of neural cells and related mechanisms[J].Med Sci Monit,2016,22:1244.
17都君君,刘洪伟.葛根素对妊娠期糖尿病模型大鼠脑组织氧化应激和细胞凋亡的影响[J].中医学报,2018,33:1407.
18孟香红,蒋建平,陈洁,等.葛根素通过抑制Calpain对抗高糖诱导的血管内皮细胞凋亡[J].中国病理生理杂志,2010,26:1992.
19杨巍,刘亚峰,周志强.葛根素对突发性耳聋患者LPO、SOD、6-Keto-PGF-1a)、TXB-2水平的影响[J].中华保健医学杂志,2018,20:144.
20王政林,蔡克万,刘文婷,等.葛根素联合神经节苷脂钠对突发性耳聋患者血液流变学及血管内皮功能的影响[J].中国药业,2018,27:60.
21刘卫红,姚琦,王陈荣,等.葛根素注射液耳后注射联合声频共振治疗老年性聋疗效观察[J].新中医,2013,45:110.