苓桂术甘汤治疗膜迷路积水豚鼠P38MAPK信号通路实验研究

Experimental Study of Linggui Zhugan Decoction on P38MAPK Signal Pathway in Guinea Pigs with Membranous Labyrinthine Hydrops

目的探索P38MAPK信号通路与苓桂术甘汤治疗膜迷路积水豚鼠的关联性。方法随机将36只健康红目豚鼠随机分为空白组、模型组、苓桂组及苓桂加α-LA组,每组各9只豚鼠(18耳),除空白组外,其余3组采用腹腔注射醋酸去氨加压素方法造模,6μg·kg^-1·d^-1,连续注射10 d。造模成功后,苓桂组豚鼠每日予苓桂术甘汤2.79 g/kg灌胃,苓桂加α-LA组每日予2.79 g/kg及α-LA 46.5 mg/kg同时灌胃,空白组及模型组以1 mL生理盐水灌胃。各组正常饲养,7 d后取材。采用HE染色法观察各组豚鼠耳蜗积水情况,免疫组化、荧光定量PCR方法以及Western-Blot法观察P38MAPK、p-P38MAPK、ATF-2蛋白在各组豚鼠耳蜗的表达情况。选用SPSS19.0软件统计分析各实验结果,探讨P38MAPK信号通路与苓桂术甘汤治疗梅尼埃病的关联性。结果蜗管面积与耳蜗总面积比值:模型组、苓桂组、苓桂加α-LA组与空白组比较均增加(P<0.01);苓桂组与模型组比较减小(P<0.01);苓桂加α-LA组与模型组比较差异无统计学意义(P>0.05);苓桂加α-LA组比苓桂组增加(P<0.01)。免疫组化方法观察P38MAPK、p-P38MAPK、ATF-2蛋白在豚鼠耳蜗表达情况:与空白组比较,模型组、苓桂组、苓桂+α-LA组P38MAPK、p-P38MAPK、ATF-2蛋白表达均下调(P<0.01);与模型组比较,苓桂组P38MAPK、p-P38MAPK、ATF-2蛋白以及苓桂+α-LA组p-P38MAPK蛋白表达水平上调(P<0.01);与苓桂组比较,苓桂+α-LA组P38MAPK、p-P38MAPK、ATF-2蛋白表达均下调(P<0.01)。荧光定量PCR方法观察P38MAPK mRNA/p-P38MAPK mRNA、ATF-2 mRNA在豚鼠耳蜗表达情况:与空白组比较,模型组、苓桂组P38MAPK mRNA/p-P38MAPK mRNA、ATF-2 mRNA表达水平下调(P<0.01),苓桂+α-LA组ATF-2 mRNA表达水平下调(P<0.01);与模型组比较,苓桂组、苓桂+α-LA组P38MAPK mRNA/p-P38MAPK mRNA、苓桂组ATF-2 mRNA表达水平上调(P<0.01);与苓桂组比较,苓桂+α-LA组P38MAPK mRNA/p-P38MAPK mRNA、ATF-2 mRNA表达下调(P<0.01)。Western-Blot方法观察P38MAPK、p-P38MAPK、ATF-2蛋白在耳蜗表达情况:与空白组比较,模型组、苓桂组、苓桂+α-LA组P38MAPK、p-P38MAPK、ATF-2蛋白表达均下调(P<0.01);与模型组比较,苓桂组、苓桂+α-LA组P38MAPK、p-P38MAPK、ATF-2蛋白表达水平上调(P<0.01);与苓桂组比较,苓桂+α-LA组P38MAPK、p-P38MAPK、ATF-2蛋白表达均下调(P<0.01)。结论苓桂术甘汤可在一定程度上减轻豚鼠膜迷路积水。苓桂术甘汤治疗梅尼埃病可能是通过P38MAPK信号通路实现的。α-LA可能抑制P38MAPK通路的活性,进而减少氧化应激反应的发生。

Objective To explore the relationship between P38MAPK signal pathway and Linggui Zhugan Decoction in the treatment of membranous hydrops in guinea pigs.Methods 36 healthy red eye guinea pigs were randomly divided into three groups:blank group,model group,Linggui group and Linggui plusα-LA group.Each group had 9 guinea pigs(18 ears)each.Except blank group,the other three groups were injected intraperitoneally with 6μg·kg^-1·d^-1 deammopressin acetate for 10 days.After the model was established successfully,the rats in Linggui group were given 2.79 g/kg Linggui Zhugan Decoction daily,the rats in Linggui plusα-LA group were given 2.79 g/kg and 46.5 mg/kgα-LA at the same time,the rats in blank group and model group were given 1 mL normal saline.All groups were fed normally and collected 7 days later.The expression of p38MAPK,P-P38MAPK and ATF-2 in the cochlea of each group was observed by HE staining,immunohistochemistry,fluorescence quantitative PCR and Western blot.Spss 19.0 software was used to analyze the experimental results,and to explore the relationship between P38MAPK signal pathway and Linggui Zhugan Decoction in the treatment of Meniere’s disease.Results The ratio of cochlear duct area to total cochlear area:compared with the blank group,the model group,Linggui group,Linggui plusα-LA group increased(P<0.01);compared with the model group,Linggui plusα-LA group decreased(P<0.01);compared with the model group,Linggui plusα-LA group had no statistical difference(P>0.05);compared with Linggui group,Linggui plusα-LA group increased(P<0.01).Immunohistochemical method was used to observe the expression of p38MAPK,P-P38MAPK and ATF-2 protein in guinea pig cochlea:compared with the blank group,the expression of P38MAPK,p-P38MAPK and ATF-2 protein in model group,Linggui group and Linggui+α-LA group were all down regulated(P<0.01);compared with the model group,the expression of P38MAPK,p-P38MAPK,ATF-2 protein and P-P38MAPK protein in Linggui+α-LA group were up regulated(P<0.01).Compared with Linggui group,P38MAPK,p-P38MAPK and ATF-2 protein expression in Linggui+α-LA group were all down regulated(P<0.01).The expression of p38MAPK mRNA/P-P38MAPK mRNA and ATF-2 mRNA in guinea pig cochlea was observed by fluorescence quantitative PCR:compared with the blank group,p38MAPK mRNA/P-P38MAPK mRNA and ATF-2 mRNA in model group and Linggui group were down regulated(P<0.01),and ATF-2 mRNA in Linggui+α-LA group was down regulated(P<0.01);compared with the model group,p38MAPK mRNA/P-P38MAPK mRNA and Linggui+α-LA group were down regulated(P<0.01).Compared with Linggui group,p38MAPK m RNA/P-P38MAPK mRNA and ATF-2 mRNA in Linggui+α-LA group were down regulated(P<0.01).Western blot method was used to observe the expression of P38MAPK,p-P38MAPK and ATF-2 protein in the cochlea:compared with the blank group,the expression of P38MAPK,p-P38MAPK and ATF-2 protein in the model group,Linggui group,Linggui+α-LA group were all down regulated(P<0.01);compared with the model group,the expression of P38MAPK,p-P38MAPK and ATF-2 protein in the Linggui group,Linggui+α-LA group was up regulated(P<0.01);compared with the Linggui group,The protein expression of P38MAPK,p-P38MAPK and ATF-2 in Linggui+α-LA group were all down regulated(P<0.01).Conclusion Linggui Zhugan Decoction can alleviate hydrops of membranous labyrinth in guinea pigs to some extent.The treatment of Meniere’s disease with Linggui Zhugan Decoction may be achieved through p38MAPK signal pathway.α-LA may inhibit the activity of p38MAPK pathway and reduce the occurrence of oxidative stress.