消痔灵注射液介导TGF-β1/Smad信号治疗放射性直肠炎出血的分子机制研究

Molecular Mechanism of Xiaozhiling Injection in the Treatment of Radiation Proctitis-induced Hemorrhage through Regulating TGF-β1/Smad Signal Pathway

目的研究消痔灵注射液介导转化生长因子β1(TGF-β1)/Smad信号治疗放射性直肠炎(RP)出血的分子机制。方法40只SPF级大鼠分为对照组、模型组、消痔灵组、TGF-β1组、TGF-β1联合消痔灵组各8只,对照组采用蛋白质含量20%的标准饲料喂养,其他四组6 MV X射线固定照射,制作RP大鼠模型,消痔灵组在模型制作后采用消痔灵注射液灌肠(照射后第12周),TGF-β1组在模型制作第4周开始腹腔注射10 mg SB-431542,TGF-β1联合消痔灵组在模型制作后腹腔注射10 mg SB-431542(照射后第4周)及采用消痔灵注射液灌肠(照射后第12周)。干预结束后2周,经八联试纸检测分析大鼠便血疗效,随机从中各抽取5只采集腹主动脉血标本5 m L,用于炎性因子[白细胞介素2(IL-2)、白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)]水平检测,处死,自距肛门2 cm以上获取直肠组织约2 cm,平均分成两部分,一部分放在10%的甲醛固定液中,用于HE染色,另一部分-80℃冻存,用于检测TGF-β1、Smad 2/3蛋白表达情况。结果模型组直肠组织TGF-β1、Smad 2、Smad 3蛋白表达量及血清IL-2、IL-6、TNF-α水平显著高于对照组(P<0.05);消痔灵组、TGF-β1组、TGF-β1联合消痔灵组直肠组织TGF-β1、Smad 2、Smad 3蛋白表达量及血清IL-2、IL-6、TNF-α水平显著低于模型组(P<0.05);TGF-β1联合消痔灵组直肠组织TGF-β1、Smad 2、Smad 3蛋白表达量及血清IL-2、IL-6、TNF-α水平显著低于消痔灵组、TGF-β1组(P<0.05);消痔灵组直肠组织TGF-β1、Smad 2、Smad 3蛋白表达量及血清IL-2、IL-6、TNF-α水平与TGF-β1组比较无统计学意义(P>0.05)。消痔灵组、TGF-β1组、TGF-β1联合消痔灵组大鼠便血疗效分别为75.00%、50.00%、87.50%,组间比较无统计学差异(P>0.05)。结论消痔灵注射液治疗RP出血主要通过介导TGF-β1/Smad信号通路发挥作用。

Objective To investigate the molecular mechanism of Xiaozhiling injection in the treatment of rectal hemorrhage secondary to radiation proctitis(RP)through regulating transforming growth factor-β1/Smad(TGF-β1/Smad)signal pathway.Methods 40 SPF rats were divided into control group,model group,Xiaozhiling injection group,TGF-β1 inhibitor group and TGF-β1 inhibitor+Xiaozhiling injection group,each with 10 rats.Rats in control group were fed with standard diet containing 20%protein.RP models of other four groups were induced by superhigh dose of 6 MV X-rays in vitro.After the model was made,the Xiaozhiling group was given Xiaozhiling injection by gavage(12 weeks after irradiation),and the TGF-β1 group was given intraperitoneal injection of 10 mg SB-431542,TGF-β1 combined with Xiaozhiling on the 4th week after the model was made.In the group,10 mg SB-431542 was intraperitoneally injected(4th week after irradiation)and Xiaozhiling injection was given to the stomach(12th week after irradiation).Two weeks after the intervention,various indexes were detected.The efficacy was tested by uritest 8 V.5 mL of abdominal aortic blood samples were collected from 5 rats in each group,and were used to detect levels of inflammatory factors[interleukin-2(IL-2),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α)].Rectal tissue about 2 cm away from the anus were obtained,and evenly divided into two parts.One part was placed in 10%formaldehyde fixed solution for HE staining,and the other part was frozen at-80℃to detect the expression of TGF-β1 and Smad 2/3.Results The protein expression of TGF-β1,Smad 2 and Smad 3 in rectal tissue of model group was significantly higher than that of control group(P<0.05);the protein expression of TGF-β1,Smad 2 and Smad 3 in rectal tissue of Xiaozhiling group was significantly lower than that of model group(P<0.05);the protein expression of TGF-β1,Smad 2 and Smad 3 in rectal tissue of TGF-β1 group was significantly lower than that of model group(P<0.05)The expression of TGF-β1,Smad 2 and Smad 3 in rectal tissue of Xiaozhiling group was not significantly different from that of TGF-β1 group(P>0.05)The protein expression of TGF-β1,Smad 2,Smad 3 and serum IL-2,IL-6,TNF-αlevels in Xiaozhiling group,TGF-β1 group,TGF-β1 combined with Xiaozhiling group were significantly lower than those in model group(P<0.05);the protein expression of TGF-β1,Smad 2,Smad 3 and serum IL-2,IL-6,TNF-αlevels in Xiaozhiling group,TGF-β1 combined with Xiaozhiling group were significantly lower than those in model group(P<0.05)The protein expression of TGF-β1,Smad 2,Smad 3 and serum IL-2,IL-6,TNF-αlevels in Xiaozhiling group were significantly lower than those in Xiaozhiling group and TGF-β1 group(P<0.05);the protein expression of TGF-β1,Smad 2,Smad 3 and serum IL-2,IL-6,TNF-αlevels in Xiaozhiling group were not significantly different from those in TGF-β1 group(P>0.05).The clinical efficacy of Xiaozhiling injection group,TGF-β1 inhibitor group and TGF-β1 inhibitor+Xiaozhiling injection group were 75.00%、50.00%、87.50%,with no statistic difference among groups(P>0.05).Conclusion Xiaozhiling injection plays an important role in the treatment of hemorrhage secondary to RP by mediating TGF-β1/Smad signaling pathway.