参榆洗液对痔术后大鼠痛觉敏化及cAMP/PKA信号通路和TRPV1蛋白表达的影响

Effects of Shenyu washing solution on pain sensitization and expressions of cAMP PKA signaling pathway and TRPV1 in rats after hemorrhoids surgery

目的探讨参榆洗液对痔术后大鼠痛觉敏化及环磷酸腺苷/蛋白激酶A(cAMP/PKA)信号通路、香草酸瞬时受体亚型1(TRPV1)蛋白表达的影响。方法取40只雄性SD大鼠,随机选择其中8只作为空白组,其余大鼠采用冰醋酸建立痔术后创面模型。将造模成功大鼠随机分为模型组及参榆洗液低、中、高剂量组,每组8只。自成功造模后的第1天开始,模型组给予高压灭菌水局部熏洗,参榆洗液低、中、高剂量组分别予以参榆洗液0.4 g生药/mL、0.8 g生药/mL、1.6 g生药/mL局部熏洗,均2次/d,每次15 min,2次治疗间隔12 h,连续7 d。记录大鼠第1,4,7天换药刺激后3 min内首次舔舐肛周创面潜伏时间、扭体反应次数以及舔舐肛周创面总时间,观察造模后和干预3,5,7 d后创面情况并记录创面愈合率,HE染色观察干预7 d后创面组织病理学形态,Western blot法检测干预7 d后创面组织中cAMP、PKA蛋白和背根神经节中TRPV1、p-PKA蛋白表达情况。结果与空白组比较,模型组大鼠首次舔舐肛周创面潜伏时间明显缩短(P<0.05),扭体反应次数明显增加(P<0.05),舔舐肛周创面总时间明显延长(P<0.05);创面组织中有大量中性粒细胞与淋巴细胞浸润,组织水肿明显;创面组织中cAMP、PKA蛋白相对表达量及背根神经节中TRPV1、p-PKA蛋白相对表达量均明显升高(P均<0.05)。与模型组比较,参榆洗液中、高剂量组大鼠首次舔舐肛周创面潜伏时间明显延长(P均<0.05),扭体反应次数明显减少(P均<0.05),舔舐肛周创面总时间明显缩短(P均<0.05);参榆洗液各组干预5,7 d后创面愈合率更高(P均<0.05);参榆洗液各组大鼠创面组织中中性粒细胞、淋巴细胞浸润减少,成纤维细胞、新生血管增多;参榆洗液各组创面组织中cAMP、PKA蛋白相对表达量及背根神经节中TRPV1、p-PKA蛋白相对表达量均明显降低(P均<0.05),且呈剂量依赖性下降(P均<0.05)。结论参榆洗液可呈剂量依赖性改善痔术后大鼠痛觉敏化,促进创面愈合,机制可能与下调cAMP/PKA信号通路相关蛋白及TRPV1蛋白表达有关。

Objective It is to investigate the effects of Shenyu washing solution on pain sensitization and expressions of cyclic adenosine monophosphate/protein kinase A(cAMP/PKA)signaling pathway and tansient receptor potential vanilloid 1(TRPV1)proteins in rats after hemorrhoidal surgery.Methods Forty male SD rats were taken,8 of which were randomly selected as the blank group,and the rest of the rats were modeled with glacial acetic acid to establish models of postoperative hemorrhoidal trauma.The successfully modeled rats were randomly divided into model group and the low,medium and high dose groups of Shenyu washing solution,with 8 rats in each group.Starting from the first day after successful modeling,the model group was given autoclaved water by local fumigation,and the low,medium,and high dose groups of Shenyu washing solution were given Shenyu washing solution 0.4 g crude drug/mL,0.8 g crude drug/mL,and 1.6 g crude drug/mL by local fumigation,respectively,all twice daily,15 min per time,and the interval was 12 h between two treatments,continuously treated for 7 days.The latency time of first licking the perianal wound,the times of torsion reactions and the total time of licking perianal wound within 3 min after drug dressing stimulation were recorded in the rats on the 1 st,4 th and 7 th days,the wound status after modeling and 3,5,7 days of intervention were observed,and the wound healing rates were calculated,the histopathological morphology of the wounds after 7 days of intervention was observed by HE staining,the expressions of cAMP and PKA proteins in traumatic tissue and TRPV1 and p-PKA proteins in dorsal root ganglion after 7 days of intervention were detected by Western blot method.Results Compared with the blank group,the latency time of first licking the perianal wound in rats of the model group was significantly shortened(P<0.05),the times of torsion reactions and the total time of licking perianal wound were significantly increased(all P<0.05);there were a large number of neutrophils and lymphocytes infiltrating in the traumatized tissues,and the tissue edema was obvious;the relative expressions of cAMP and PKA proteins in traumatic tissue and the relative expressions of TRPV1 and p-PKA in dorsal root ganglion were significantly increased(all P<0.05).Compared with the model group,the latency time of first licking the perianal wound in rats of the middle and high dose groups of Shenyu washing solution were significantly prolonged(P<0.05),the times of torsion reactions and the total time of licking perianal wound were significantly decreased(all P<0.05);the healing rate of perianal wounds was higher in each dose groups of Shenyu washing solution after 5,7 days of intervention(all P<0.05).The infiltration of neutrophil and lymphocyte was decreased and fibroblasts and neovascularization was increased in traumatic tissues of rats in all groups;the relative expression of cAMP and PKA proteins in the traumatic tissues of each dose groups of Shenyu washing solution,and the relative expressions of cAMP and PKA proteins in traumatic tissue and that of TRPV1 and p-PKA in dorsal root ganglion were all significantly decreased(all P<0.05),and the decreases were dose-dependent.Conclusion Shenyu washing solution can dose-dependently improve pain sensitization and promote wound healing in rats after hemorrhoidal surgery,and the mechanism may be related to down-regulating cAMP/PKA signaling pathway-related proteins and TRPV1 protein expression.