泻火化瘀通窍法对耳蜗微循环障碍毛细胞凋亡的实验研究

Experimental study on the effects of fire-draining,stasis-transforming and orifice-freeing methods on apoptosis of hair cells induced by microcirculation disorder in the cochlear of guinea pigs

目的探讨泻火化瘀通窍法改善豚鼠耳蜗微循环障碍的作用机制。方法健康豚鼠70只,随机分成,即正常组、模型组、行气组、活血化瘀组、泻火化瘀通窍组,每组14只。除正常组外,其余各组分别进行光化学诱导法造模,然后各组分别予0.9%生理盐水、0.9%生理盐水、行气中药液、活血化瘀中药液、泻火化瘀通窍中药液灌胃。每日2次,连续10天。取耳蜗组织,实时荧光定量PCR检测Bcl-2 mRNA及Bax mRNA,Western blot方法检测Bcl-2及Bax蛋白。结果 Bcl-2 mRNA活性水平依次为泻火化瘀通窍组>活血化瘀组>行气组>模型组(P<0.05);Bax mRNA活性水平依次为泻火化瘀通窍组<活血化瘀组<行气组<模型组。Bcl-2/Bax蛋白比值依次为泻火化瘀通窍组>活血化瘀组>行气组>模型组。结论泻火化瘀通窍药和活血化瘀药均能抑制Bcl-2和Bax活性改变诱导的豚鼠耳蜗细胞凋亡,尤以泻火化瘀通窍药效优。

Objective To explore the mechanisms of fire-draining, stasis-transforming and orifice-freeing methods to improve microcirculation disorder experimentally induced in the cochlear of guinea pigs. Method Seventy healthy guinea pigs were randomly divided into 5 groups, i.e. blank group （BG）, modeling group （MG）, Qi-moving group （QMG）, blood-quickening stasis-transforming group （BSG） and fire-draining stasis-transforming orifice-freeing group （FSOG）, with 14 in each group. All animals were prepared as model of microcirculation disorder in their cochlea by photochemical inducing method, other than those in BG. Then, guinea pigs in BG, MG, QMG, BSG and FSOG were given 2 ml of 0.9% normal saline, herbal decoctions of Qi-moving, blood-quickening stasis-transforming, or fire-draining stasis-transforming orifice-freeing respectively, twice a day for 10 days, by intragastric administration. By the end of feeding period, all guinea pigs were put into death to collect cochlear specimens for Bcl-2 and Bax determination by qRT-PCR for mRNA and Western-blotting for protein respectively. At last, a comparative analysis was made to explore their differences among different groups. Result The expressive levels of Bcl-2 mRNA were FSOG, BSG, QMG and MG in the order from higher level to lower level, while that of Bax mRNA were seen in a reversed order to that of Bcl-2 mRNA, i.e. MG, QMG, BSG and FSOG, also in the order from higher to lower. The order of Bcl-2/Bax ratio was ranged as the same way as that of Bcl-2 mRNA, i.e. FSOG, BSG, QMG and MG in the order from higher level to lower level ratio. Conclusion Herbs with the effects of fire-draining stasis-transforming orifice-freeing and those with the effects of blood-quickening stasis-transforming both can inhibit apoptosis in cochlear hair cells induced by microcirculation disorder through regulating expressive activities of Bcl-2 and Bax genes. This kind of effect is especially superior for those with the effects of fire-draining, stasis-transforming and orifice-freeing.