痛泻要方对肝郁脾虚型溃疡性结肠炎模型大鼠结肠SERT,肝脏5-HT_(2A)R蛋白表达的影响

Effect of Tongxie Yaofang on Expressions of Colon SERT and Liver 5-HT_(2A)R Proteins in Rats with Ulcerative Colitis Model of Liver Stagnation and Spleen Deficiency

目的:观察痛泻要方对肝郁脾虚型溃疡性结肠炎(UC)模型大鼠结肠5-羟色胺转运体(SERT),肝脏5-羟色胺(5-HT)2A受体(5-HT_(2A)R)蛋白及血清5-HT,炎症因子等表达的影响,探讨肝郁脾虚证候基础及痛泻要方干预机制。方法:将50只SD雄性大鼠随机分为正常组,模型组,痛泻要方高、中、低剂量组(10,5,2.5 g·kg^(-1)),柳氮磺嘧啶组(0.3 g·kg^(-1))。采用2,4,6-三硝基苯磺酸(TNBS)/乙醇溶液灌肠+束缚应激+饮食失节建立肝郁脾虚型溃疡性结肠炎模型。造模成功后,给予相应药物干预21 d后取材,采用苏木素-伊红(HE)染色,油红O染色分别观察各组大鼠结肠、肝脏病理学改变。酶联免疫吸附测定(ELISA)检测大鼠血清白细胞介素(IL)-6,IL-9,5-HT,超氧化物歧化酶(SOD)水平,蛋白免疫印迹法(Western blot)检测大鼠结肠SERT,肝脏5-HT_(2A)R蛋白表达。结果:与正常组比较,模型组结肠形成明显溃疡,肝脏脂滴形成增加。血清IL-6,IL-9,5-HT水平上升,SOD水平明显下降(P<0.05),结肠SERT蛋白表达量显著下降,肝脏5-HT_(2A)R蛋白表达量明显上升(P<0.05);与模型组比较,痛泻要方高、中剂量组及柳氮磺嘧啶组结肠病理损伤得到改善,肝脏脂滴形成减少。痛泻要方各剂量组及柳氮磺嘧啶组血清IL-6,IL-9,5-HT水平均下降,SOD水平明显上升(P<0.05)。痛泻要方高、低剂量组及柳氮磺嘧啶组结肠SERT蛋白表达量上升,痛泻要方中、低剂量组及柳氮磺嘧啶组肝脏5-HT_(2A)R蛋白表达量明显下降(P<0.05)。结论:痛泻要方可能通过上调结肠SERT的表达,从而降低5-HT的含量,调节肠道动力及感觉系统;抑制IL-6,IL-9等炎性因子的表达,发挥抗炎作用;降低5-HT含量及肝脏5-HT_(2A)R表达,升高SOD水平,调节情志及肝脏脂质代谢,发挥对肝郁脾虚型溃疡性结肠炎干预作用。

Objective:To observe the effect of Tongxie Yaofang on the expressions of colon serotonin transporter(SERT),liver 5-hydroxytryptamine2Areceptor(5-HT_(2A)R)protein,serum 5-HT and inflammatory factors in ulcerative colitis(UC)model rats of liver stagnation and spleen deficiency,in order to explore the basis of syndrome of liver stagnation and spleen deficiency and the intervention mechanism of Tongxie Yaofang.Method:Fifty male SD rats were randomly divided into blank control group,model group,high,medium and low-dose Tongxie Yaofang group(10,5,2.5 g·kg^(-1)),and salazosulacil group(0.3 g·kg^(-1)).The ulcerative colitis model of liver depression and spleen deficiency was established by 2,4,6-trinitrobenzene sulfonic acid(TNBS)/ethanol solution enema+restraint stress+diet loss.After successful modeling,the samples were collected after21 days of drug intervention.Htoxylin eosin(HE)staining and oil red staining were used to observe the pathological changes of colon and liver in each group.Serum interleukin-6(IL-6),IL-9,5-HT and superoxide dismutase(SOD)were detected by enzyme linked immunosorbent assay(ELISA).Protein expressions of SERT in the colons and 5-HT_(2A)R in liver of rats were detected by Western blot.Result:Compared with the normal group,obvious ulcers were formed in the colon and lipid droplets in the liver increased in the model group,serum levels of IL-6,IL-9 and 5-HT in the model group increased,while the level of SOD decreased(P<0.05).The protein expression of SERT in colon decreased,whereas the protein expression of 5-HT_(2A)R in liver increased(P<0.05).Compare with model group,the pathological damage of colon was improved,and the formation of lipid droplets in liver was reduced in high,medium-dose Tongxie Yaofang groups and sulfasalazine group.The serum levels of IL-6,IL-9 and 5-HT decreased,while the level of SOD increased in Tongxie Yaofang group and sulfasalazine group(P<0.05).The protein expression of SERT in colon increased in high,low-dose Tongxie Yaofang groups and sulfasalazine group,and the protein expression of 5-HT_(2A)R in liver decreased in medium,low dose Tongxie Yaofang groups and sulfasalazine group(P<0.05).Conclusion:Tongxie Yaofang may reduce the content of 5-HT,and regulate the intestinal motility and sensory system by up-regulating the expression of SERT in the colon,inhibit the expressions of IL-6,IL-9 and other inflammatory factors,and play an antiinflammatory role,reduce the content of 5-HT and the expression of 5-HT_(2A)R in the liver,increase the level of SOD,regulate emotion and lipid metabolism in the liver,and then exert the intervention effect on ulcerative colitis with liver depression and spleen deficiency on the whole.