基于PLC/IP3通路探讨岩黄连对新生儿高胆红素血症大鼠听觉系统损伤的影响

Effects of Corydalis saxicola on auditory system damage in rats with neonatal hyperbilirubinemia based on PLC/IP3 pathway

目的探讨岩黄连Corydalis saxicola对新生儿高胆红素血症(neonatal hyperbilirubinemia,NH)大鼠磷脂酶C(phospholipase C,PLC)/磷酸肌醇(inositoltriphosphate,IP3)通路及听觉系统损伤的影响。方法取7d龄SD大鼠50只,用随机数字表法分为对照组、模型组及岩黄连低、中、高剂量(40、80、160mg/kg)组,每组10只;除对照组ip生理盐水外,其余各组大鼠均按200μg/g的剂量ip胆红素建立NH大鼠模型。造模后,对照组和模型组ig给予10 mL/kg的生理盐水,岩黄连各剂量组ig给予相应剂量的岩黄连提取物,各组均给药1 d,3次/d。末次给药2 h后,观察大鼠一般行为状况,检测脑干听觉诱发电位(auditory brainstem response,ABR)和血清及脑干组织胆红素含量;HE染色观察耳蜗核组织病理变化;免疫组化染色法观察耳蜗核组织钙素(calcium,CaM)活性;蛋白免疫印迹法(Western blotting)检测脑干组织磷酸化PLC(p-PLC)、IP3蛋白表达水平。结果与对照组相比,模型组大鼠出现烦躁等神经异常行为,耳蜗核组织可见神经细胞肿胀等病理损伤,ABR阈值、耳蜗核组织CaM阳性表达率、血清及脑干组织胆红素含量升高(P<0.05),Ⅰ、Ⅱ、Ⅲ波潜伏期明显延长(P<0.05),脑干组织p-PLC、IP3蛋白表达降低(P<0.05);与模型组相比,岩黄连低、中、高剂量大鼠上述神经异常行为及耳蜗核组织病理损伤得到缓解,ABR阈值、耳蜗核组织CaM阳性表达率、血清及脑干组织胆红素含量降低(P<0.05),Ⅰ、Ⅱ、Ⅲ波潜伏期明显缩短(P<0.05),脑干组织p-PLC、IP3蛋白表达升高(P<0.05),且岩黄连发挥的作用呈剂量相关性。结论岩黄连可激活NH大鼠耳蜗核组织PLC/IP3通路蛋白表达,减轻胆红素对耳蜗核听力神经系统的毒害,改善NH大鼠听觉。

Objective To investigate the effects of Yanhuanglian(Corydalis Saxicola) on phospholipase C(PLC)/inositol phosphate(IP3) pathway and auditory system damage in neonatal hyperbilirubinemia(NH) rats. Methods A total of 50 7-d-old SD rats were randomly divided into control group, NH model group(NH group), C. saxicola low(40 mg/kg), middle(80 mg/kg) and high(160 mg/kg) dose groups, with 10 rats in each group;Except the control group, which was intraperitoneally injected with normal saline, the rats in the other groups were intraperitoneally injected with bilirubin at the dose of 200 μg/g to establish NH rat model. After modeling, the control group and NH group were given 10 mL/kg normal saline by gavage, and C. saxicola groups were given the corresponding dose of C. saxicola extract by gavage, and each group was given medicine for 1 d, 3 times a day. Two hours after the last administration, the general behavior and auditory brainstem response(ABR) were observed;The content of bilirubin in serum and brain stem tissue was detected by absorbance(A450);HE staining was used to observe the pathological changes of cochlear nucleus;The activity of calcium(CaM) in cochlear nucleus was observed by immunohistochemistry;Moreover, Western blot was used to detect the expression levels of p-phospholipase C(PLC) and IP3 in brain stem. Results Compared with those in control group, the rats in NH group had abnormal nervous behaviors such as irritability, and pathological damage such as nerve cell swelling in cochlear nucleus tissue, ABR threshold, CaM positive expression rate in cochlear nucleus and bilirubin content in serum and brain stem tissue were higher(P < 0.05), the latencies of waves Ⅰ, Ⅱ and Ⅰ were significantly prolonged(P < 0.05), and the expressions of p-PLC and IP3 proteins in brain stem tissue were lower(P < 0.05);Compared with those in NH group, the above abnormal behaviors and pathological damage of cochlear nucleus in C. saxicola low, middle and high dose groups were alleviated, ABR threshold, CaM positive expression rate in cochlear nucleus and bilirubin content in serum and brain stem tissue were lower(P < 0.05), the latencies of waves Ⅰ, Ⅱ and Ⅲ were significantly shortened(P < 0.05), and the expressions of p-PLC and IP3 proteins in brain stem tissue were higher(P < 0.05), and the C. saxicola group exerted the effect in a dose dependent manner. Conclusions C. saxicola can activate the expression of PLC/IP3 pathway protein in cochlear nucleus of NH rats, reduce the toxicity of bilirubin to the auditory nervous system of cochlear nucleus, and improve the hearing of NH rats.