柴胡疏肝散对糖尿病周围神经病变大鼠瞬时受体电位香草酸亚型1/降钙素基因相关肽通路及坐骨神经电生理变化的影响

Effects of Chaihu Shugan powder on TRPV1/CGRP pathway and electrophysiological changes of sciatic nerve in DPN rats

目的探究柴胡疏肝散对糖尿病周围神经病变(DPN)大鼠瞬时受体电位香草酸亚型1(TRPV1)/降钙素基因相关肽(CGRP)通路及坐骨神经电生理变化的影响。方法2019年12月至2020年9月,从北京生命科学研究所动物实验中心购入SD大鼠,采用高脂饲料+小剂量链脲佐菌素(STZ)腹腔注射法建立DPN大鼠模型,分为对照组、模型组、弥可保组(175μg/kg)、低中高剂量柴胡疏肝散(3.15、6.30、12.60 g/kg),每组5只,造模成功后,连续给药8周。8周末检测各组大鼠摆尾温度阈值,肌电图仪检测大鼠运动神经传导速度(MNCV)及其支配肌复合运动动作电位的波幅(AMP)、潜伏期(LAT)的变化。实时荧光定量PCR(qRT-PCR)法检测血清TRPV1、CGRP、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)mRNA的水平;苏木精-伊红(HE)染色观察坐骨神经组织病理变化;蛋白质印迹法检测坐骨神经组织中TRPV1、CGRP、TNF-α、IL-1β蛋白水平。结果与对照组[(33.15±1.28)s、(1.37±0.06)ms、(41.48±5.36)m/s、(12.32±1.55)mV]相比,模型组大鼠坐骨神经周围观察到大量浸润性巨噬细胞和单核细胞,并伴有广泛的轴突脱髓鞘,摆尾温度阈值(48.67±2.65)s、LAT(3.68±0.18)ms升高,MNCV(30.26±3.65)m/s、AMP(4.58±0.26)mV降低,血清及坐骨神经中TRPV1、CGRP、TNF-α、IL-1βmRNA或蛋白水平升高(P<0.05);与模型组相比,弥可保组、低中高剂量柴胡疏肝散组单核细胞浸润减少,脱髓鞘程度减轻,摆尾温度阈值[(37.75±1.45)s、(45.61±2.38)s、(41.74±1.68)s、(38.21±1.36)s]、LAT[(1.62±0.08)ms、(2.92±0.12)ms、(2.03±0.09)ms、(1.69±0.08)ms]降低,MNCV[(38.56±4.85)m/s、(33.63±3.21)m/s、(35.42±3.35)m/s、(38.57±4.14)m/s]、AMP[(11.95±1.02)mV、(7.61±0.75)mV、(9.35±0.92)mV、(11.89±1.03)mV]升高,血清及坐骨神经中TRPV1、CGRP、TNF-α、IL-1βmRNA或蛋白水平降低(P<0.05)。结论柴胡疏肝散可能通过抑制TRPV1/CGRP通路表达,减轻大鼠坐骨神经损伤,达到保护周围神经的作用,可能作为DPN潜在的治疗药物。

Objective To investigate the effects of Chaihu Shugan powder on transient receptor potential vanlloid 1(TRPV1)/calcitonin gene-related peptide(CGRP)pathway and electrophysiological changes of sciatic nerve in rats with diabetic peripheral neuropathy(DPN).Methods From December 2019 to September 2020,SD rats were purchased from the Animal Experiment Center of Beijing Institute of Life Sciences,and the DPN rat models were established by high-fat diet and intraperitoneal injection of low-dose streptozotocin(STZ).The rats were divided into control group,model group,micobalamin group(175μg/kg),low,medium and high-dose Chaihu Shugan powder(3.15,6.30,12.60 g/kg),with 5 rats in each group.After successful modeling,the rats were treated for 8 weeks.At the end of the 8th week,the tailflick threshold temperature was measured.The motor nerve conduction velocity(MNCV)and the amplitude(AMP)and latency(LAT)of the compound motor action potential in the innervating muscles of rats were measured by electromyography.The mRNA levels of TRPV1,CGRP,tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)were detected by real-time quantitative PCR(qRT-PCR);the histopathological changes of sciatic nerve observed by hematoxylin eosin(HE)staining;the protein levels of TRPV1,CGRP,TNF-αand IL-1βwere detected by Western blotting(WB).Results Compared with those in the control group[(33.15±1.28)s,(1.37±0.06)ms,(41.48±5.36)m/s,(12.32±1.55)mV],a large number of infiltrating macrophages and monocytes were observed around the sciatic nerve in the model group,accompanied by extensive axonal demyelination,the tailflick threshold temperature(48.67±2.65)s and LAT(3.68±0.18)ms increased,MNCV(30.26±3.65)m/s and AMP(4.58±0.26)mV decreased,the mRNA and protein levels of TRPV1,CGRP,TNF-α and IL-1β in serum and sciatic nerve were higher(P<0.05);compared with those in the model group,the monocyte infiltration and demyelination degree were decreased in methycobal group and low,medium and high dose Chaihu Shugan powder group,the tailflick threshold temperature[(37.75±1.45)s,(45.61±2.38)s,(41.74±1.68)s,(38.21±1.36)s]and LAT[(1.62±0.08)ms,(2.92±0.12)ms,(2.03±0.09)ms,(1.69±0.08)ms]decreased,MNCV[(38.56±4.85)m/s,(33.63±3.21)m/s,(35.42±3.35)m/s,(38.57±4.14)m/s]and AMP[(11.95±1.02)mV,(7.61±0.75)mV,(9.35±0.92)mV,(11.89±1.03)mV]increased,the mRNA and protein levels of TRPV1,CGRP,TNF-αand IL-1βin serum and sciatic nerve were lower(P<0.05).Conclusion Chaihu Shugan powder can alleviate injury of sciatic nerve and protect the peripheral nerve by inhibiting the expression of TRPV1/CGRP pathway,which may be a potential therapeutic drug for DPN.