抵当汤及其拆方对深静脉血栓形成大鼠高速泳动族蛋白B1影响的研究

Effects of Didang Tang and Its Disassembled Prescription on High-mobility Group Box 1 Protein in Rats with Deep Vein Thrombosis

目的:观察抵当汤及其拆方对DVT大鼠高速泳动族蛋白B1(HMGB1)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的影响。方法:将150只Wistar雄性大鼠随机分为5组,抵当汤组、大黄桃仁组、水蛭地龙组、模型组、假手术组,每组30只。采用Reyers法建立下腔静脉结扎模型,术后第1、3、7天灌胃2 h后,腹腔麻醉开腹,采用酶联免疫吸附试验(ELISA)、蛋白质免疫印迹法检测高速泳动族蛋白B1(HMGB1)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平,苏木精-伊红(HE)染色观察大鼠下腔静脉的病理变化,免疫荧光(IF)、实时逆转录PCR(Realtime RT-PCR)检测大鼠下腔静脉中的HMGB1表达。结果:模型组下腔静脉内皮细胞损伤严重并大量脱落,细胞肿胀明显,形成血栓,内膜形态不规则、管壁组织肿胀,胶原纤维增生,伴有炎症细胞浸润,抵当汤组、大黄桃仁组、水蛭地龙组等用药组内皮细胞不同程度损伤和炎症细胞浸润;模型组HMGB1荧光表达较假手术组明显增多,其余用药组HMGB1荧光表达不同程度减少;与假手术组比较,模型组大鼠的高速泳动族蛋白B1(HMGB1)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)血清水平、高速泳动族蛋白B1(HMGB1)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、蛋白表达、mRNA表达明显升高(P<0.01)。与模型组比较,抵当汤组、大黄桃仁组、水蛭地龙组等用药组HMGB1、IL-6、TNF-α血清水平、蛋白表达、mRNA表达明显降低(P<0.05,P<0.01)。抵当汤组、大黄桃仁组、水蛭地龙组等用药组比较,HMGB1、IL-6、TNF-α血清水平、高速泳动族蛋白B1(HMGB1)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、蛋白表达、mRNA表达比较差异有统计学意义(P<0.05,P<0.01),术后第1、3、7天比较,HMGB1、IL-6、TNF-α血清水平、蛋白表达、mRNA表达比较差异有统计学意义(P<0.05,P<0.01)。结论:抵当汤及其拆方通过降低DVT大鼠的HMGB1、IL-6、TNF-α表达,发挥对DVT的治疗作用。

Objective:To investigate the effects of Didang Tang and its disassembled prescription on high-mobility group box-1(HMGB1) protein,tumor necrosis factor-α(TNF-α) and Interleukin-6(IL-6) in rats with deep venous thrombosis(DVT).Methods:A total of 150 SPF grade Wistar male rats were randomly divided into 5 groups:Didang Tang group,Dahuang-Taoren group,Shuizhi-Dilong group,model group and sham operation group,with 30 rats in each group.The ligation model of the inferior vena cava was established by Reyers method.The levels of HMGB1,TNF-α and IL-6 were detected by ELISA and WB.The pathological changes of the inferior vena cava of rats were observed by HE staining.The expression of HMGB1 in the inferior vena cava of rats was detected by immunofluorescence and RT-qPCR.Results:In the model group,the endothelial cells of the inferior vena cava were seriously damaged and a large number of cells fell off,with significant cell swelling and thrombus formation.The intima was irregular in shape,the wall tissue was swollen,and collagen fibers were hyperplasia,accompanied by inflammatory cell infiltration.The endothelial cells were damaged with inflammatory cell infiltration in different degrees in medication groups.The fluorescence intensity of HMGB1 in the model group was significantly higher than that in the sham group,and the fluorescence intensity of HMGB1 in the medication groups was decreased to varying degrees.Compared with the sham operation group,the serum levels,protein expression and mRNA expression of HMGB1,IL-6 and TNF-α in the model group were significantly increased(P<0.01).Compared with the model group,the serum levels,protein expression and mRNA expression of HMGB1,IL-6 and TNF-α in each medication group were significantly decreased(P<0.05,P<0.01).There were significant differences in serum level,protein expression and mRNA expression among the treatment groups(P<0.05,P<0.01),and there were significant differences in serum level,protein expression and mRNA expression on postoperative day 1,3 and 7(P<0.05,P<0.01).Conclusion:Didang Tang and its disassembled prescription play a therapeutic effect on DVT by reducing the expressions of HMGB1,IL-6 and TNF-α in rats with DVT.