帕金森鼠结肠黏膜炎性反应与多巴胺和5-羟色胺受体表达

目的 主要观察多巴胺（dopamine，DA）和5-羟色胺（serotonin，5-HT）受体在结肠黏膜的分布，6-羟多巴（6-hydroxydopamine，6-OHDA）大鼠结肠黏膜通透性和炎性反应因子的改变以及DA和5-HT含量及其受体的表达变化，初步探讨DA与5-HT在结肠黏膜保护中的可能作用。方法 双侧黑质内注射6-OHDA制作帕金森病（Parkinson's disease，PD）大鼠模型。通过免疫荧光染色和Western blotting法对模型进行鉴定，利用高效液相色谱-电化学检测法检测DA和5-HT的含量，通过实时定量PCR和Western blotting法检测DA和5-HT受体的表达。结果 6-OHDA大鼠黑质中酪氨酸羟化酶蛋白表达水平明显降低（对照组：0.335±0.073，模型组：0.132±0.028，P<0.05），粪便含水量显著减少（对照组：0.119%±0.005%，模型组：0.062%±0.006%，P<0.01），转棒停留时间明显缩短［对照组（35.330±3.148） s，模型组（24.000±1.424） s，P<0.01］，表明造模成功。结肠黏膜电阻降低［对照组（73.750±4.024）Ω/cm²，模型组（53.400±6.282）Ω/cm²，P<0.05］，异硫氰酸荧光素（fluorescein isothiocyanate，FITC）-葡聚糖透过量显著升高［60 min时，对照组：（18.860±1.856）μg/L，模型组：（37.000±4.457）μg/L，P<0.05］，炎性反应因子肿瘤坏死因子（tumor necrosis factor-α，TNF-α）明显增加［对照组：（269.10±18.00） ng/g，模型组：（340.40±21.52） ng/g，P<0.05］，提示6-OHDA大鼠结肠黏膜存在炎性反应。进一步发现6-OHDA大鼠结肠黏膜中DA受体D₁（对照组：0.728±0.132，模型组：0.272±0.067，P<0.01）和D₅（对照组：0.721±0.036，模型组：0.543±0.051，P<0.05）的蛋白表达水平明显降低，5-HT₃的表达虽有升高趋势，差异无统计学意义（P<0.01）。而DA与5-HT含量，以及D₂和5-HT₄的表达水平均无明显变化。结论 6-OHDA大鼠结肠黏膜中DA受体D₁和D₅的表达明显降低，黏膜通透性与炎性反应因子TNF-α升高，提示6-OHDA大鼠结肠黏膜存在有炎性反应。

Inflammation and the expression of dopamine and 5-HT receptors in colonic mucosa of Parkinson's rats

Objective To report the distribution of dopamine (DA) and serotonin (5-HT) receptors in the colonic muscular layer and their potential role in the motility regulation in the Parkinson's disease (PD) model 6-hydroxydopamine (6-OHDA) rats, it is not clear their expressions in the colonic mucosa and correlation with the colonic mucosal dysfunction in the PD rats. The present study aims to investigate the distribution of DA and 5-HT receptors in the colonic mucosa, and the colonic mucosal inflammatory and permeability and the expression change of the DA and 5-HT receptors in the colonic mucosa in 6-OHDA rats. Methods Injection of 6-OHDA to the bilateral substantia nigras of rats, to make PD model (6-OHDA rats). Immunofluorescence (IF) and western blotting were used to observe the injection sites and the extent of DA neurons' injury. DA and 5-HT content was measured by high performance liquid chromatography-electrochemistry detection (HPLC-EDC). The mRNA and protein expression of DA receptors (D₁, D₂, D₅) and 5-HT receptors (5-HT₃, 5-HT₄) was detected by Real-time PCR and Western blotting. Results In the 6-OHDA rats, the protein expression of tyrosine hydroxylase (the synthetic rate-limiting enzyme of dopamine) in substantia nigra was decreased (control:0.335±0.073, model:0.132±0.028, P<0.05), fecal water content was reduced[control:(0.119±0.005)%, model:(0.062±0.006)%, P<0.01] and the time of rotarod test was shortened[control:(35.330±3.148)s, model:(24.000±1.424)s, P<0.01], indicating the model is successful. Furthermore, the colonic transepithelial resistance was decreased[control:(73.750±4.024)Ω/cm², model:(53.400±6.282)Ω/cm², P<0.05], the mucosal fluorescein isothiocyanate (FITC)-dextran permeability was increased[at 60 minutes, control:(18.860±1.856)μg/L, model:(37.000±4.457)μg/L, P<0.01], and the TNF-α was also increased[control:(269.10±18.00)ng/g, model:(340.40±21.52)ng/g, P<0.05], suggesting a higher mucosal permeability and inflammation. In the colonic mucosa of 6-OHDA rats, the expression of D₁[control:(0.728±0.132), model:(0.272±0.067), P<0.01] and D₅ (control:0.721±0.036, model:0.543±0.051, P<0.05) were decreased, but the DA and 5-HT content, and the receptors D₂, 5-HT₃ and 5-HT₄ had no significant alteration although 5-HT₃ (control:0.078±0.016, model:0.103±0.034, P>0.05) had a increase tendency. Conclusion The present study demonstrates that 6-OHDA rats manifest a reduction of D₁ and D₅ expressions in colonic mucosa, and increase in colonic mucosal permeability and TNF-α level, which suggests a colonic mucosal inflammation. However, whether the reduction of colonic D₁ and D₅ is involved in the formation of mucosal inflammation in 6-OHDA rats needs to be further investigated.