疏肝健脾方对小鼠脊髓背跟神经元TRPV1电流通道及p-TRPV1、SP、CGRP受体表达的影响

目的探讨疏肝健脾方对小鼠脊髓背跟神经元香草酸受体亚型1(the transient receptor potential vanilloiol type 1,TRPV1)电流通道及p-TRPV1、物质P(SP)、降钙素基因相关肽(CGRP)受体表达的影响。方法参照血清药理学方法,正常组及疏肝健脾方低、中、高剂量组含药血清;分离并原代培养C57小鼠脊神经后角神经节(Dorsal root ganglion,DRG)细胞,通过免疫荧光法进行细胞鉴定,DRG细胞经辣椒素(Capsaicin,CAP)干预后分组(即低、中、高剂量组)予含药血清干预,利用全细胞膜片钳技术记录的DRG细胞TRPV1通道电流;采用RT-PCR及Western-blot检测小鼠DRG神经元中p-TRPV1、SP、CGRP表达变化。结果经细胞鉴定,证实成功分离小鼠DRG细胞;DRG细胞形态呈圆形,胞体较小,突起较长;结肠平滑肌细胞形态细胞形状椭圆形,束状排列,密集与稀疏处相互交错呈"峰谷"状。疏肝健脾方无法直接激活DRG神经元中的p-TRPV1通道,却可正向调控辣椒素激动的p-TRPV1电流;低、中、高剂量组DRG细胞内p-TRPV1、CGRP的mRNA及蛋白表达量随药物浓度增高逐步下降(P<0.05),SP表达mRNA及蛋白表达量随药物浓度增高逐步上升(P<0.05)。结论疏肝健脾方可通过下调p-TRPV1表达,从而抑制CGRP的表达,上调SP表达,发挥防治肠易激综合征(Irritable Bowel Syndrome,IBS)的效用。

Effects of Shugan Jianpi Recipe on TRPV1 Current Channel and Expressions of p-TRPV1, SP and CGRP Receptors in Spinal Dorsal Horn Neurons of Mice

Objective To explore the effect of Shugan Jianpi Recipe on the expressions of TRPV1 current channel and p-TRPV1,SP and CGRP receptors in spinal cord heel neurons of mice.Methods According to the serum pharmacology method,there were normal group and drug-containing serum of Shugan Jianpi Recipe low,medium and high dose groups.We isolated and primarily cultured C57 mice DRG(dorsal root ganglion)cells.By immunofluorescence cell identification,DRG cells were treated with Capsaicin(CAP)intervention(low,medium and high dose groups)with drug-containing serum intervention,and DRG1(the transient receptor potential vanilloid type)was recorded by whole-cell patch clamp technique 1)channel current.RT-PCR and Western-blot were used to detect the expression changes of p-TRPV1,SP and CGRP in mouse DRG neurons.Results After cell identification,it was confirmed that mice DRG cells were successfully isolated.The morphology of DRG cells was round,the cell body was small,and the processes were long.The shape of colonic smooth muscle cells was elliptical,bundledand dense and sparse.Shugan Jianpi Recipe can not only directly activate p-TRPV1 channel in DRG neurons,but also positively regulate capsaicin-activated p-TRPV1 current.The mRNA and protein expressions of P-TRPV1 and CGRP in DRG cells of low,medium and high dose groupsdecreased gradually with the increase of drug concentration(P<0.05),and the expressions of SP and mRNA increased gradually with the increase of drug concentration(P<0.05).Conclusion Shugan Jianpi Recipe can inhibit the expression of CGRP and up-regulate the expression of SP by down-regulating the expression of p-TRPV1,and plays a role in the prevention and treatment of irritable bowel syndrome.