肝豆扶木汤对Wilson病患者血清TGF-β1、TNF-α、IL-6的影响及对TX小鼠肝纤维化的研究

发布时间：2018-08-11 19:09

【摘要】：目的:(1)以年龄、病程无差异的Wilson病(WD)肝纤维化患者作为研究对象,选用丹参颗粒作为对照治疗方法,观察肝豆扶木汤(GDFMT)对WD肝纤维化患者UWDRS肝脏功能评分、中医证候积分值、TGF-β1、TNF-α、IL-6值的影响,评价GDFMT治疗WD肝纤维化患者的临床疗效及对相关生化指标的影响。(2)以TGF-β1/smad信号转导通路为基础,以TX小鼠作为实验观察对象,应用可多靶点治疗WD肝纤维化的肝豆扶木汤(GDFMT)为干预药物,经过高、中、低浓度GDFMT不同疗程干预后,采用半定量RT-PCR检测肝组织中TGF-β1、TβRI、TβRII、Smad2、Smad3、Smad4和Smad7,探讨GDFMT抗WD肝纤维化的疗效及其通路机制。方法:临床部分:将符合WD肝纤维化纳入标准的60例患者随机分为治疗组(GDFMT组)30例,对照组(丹参组)30例。治疗组治疗药物予以GDFMT,对照组治疗药物予以丹参颗粒,同时予以2组患者相同的驱铜补钙基础治疗,基础治疗以8天作为一个疗程,予以DMPS静脉驱铜治疗6天,后2天予以补钙治疗,总计4个疗程。评价两组患者治疗前后UWDRS肝脏功能积分值、中医证候积分值;并检测两组患者TGF-β1、TNF-α、IL-6指标变化。实验部分:192只雄性TX小鼠被随机分为以下6组:GDFMT高剂量组、GDFMT中剂量组、GDFMT低剂量组(分别简称“高剂量组、中剂量组、低剂量组”)、模型组(生理盐水)、青霉胺组(青霉胺)、丹参组(丹参);另外以DL小鼠32只为正常对照组,共7组。予以7组受试动物相应的药物处理,每天灌胃1次,连续56天,分别于灌胃治疗后2、4、6、8周结束时将受试动物分批处死,每组每次8只。采用半定量RT-PCR检测各周各批TX小鼠肝组织内TGF-β1、TβRI、TβRII、Smad2、Smad3、Smad4和Smad7的m RNA相对表达量,并作出比较。结果:(1)临床部分:临床总疗效:治疗4个疗程后,可见治疗组临床总疗效明显优于对照组(P0.01);治疗前两组患者中医证候积分值比较无差异(P0.05);两组患者在治疗前UWDRS肝脏功能评分值无差异(P0.05);两组分别与同组治疗前相比,治疗后中医证候积分值均降低(P0.01,P0.05);与同组治疗前相比,2组治疗后UWDRS肝脏功能评分值均降低,具有统计学意义(P0.01,P0.05);2组患者治疗后中医证候积分值对比,治疗组较对照组降低更明显(P0.01);2组患者治疗后UWDRS肝脏功能评分值对比,治疗组治疗效果优于对照组(P0.01)。临床疗效性指标评估:血清TGF-β1、TNF-α、IL-6治疗前两组间比较,治疗组和对照组治疗前TGF-β1、TNF-α、IL-6值均无差异(P0.05);两组疗前、疗后组内比较,治疗组疗后较疗前TGF-β1、TNF-α、IL-6均显著降低(P0.01),对照组治疗后TGF-β1、TNF-α、IL-6较治疗前有明显下降(P0.01,P0.05);治疗组、对照组治疗后比较,治疗组比对照组改善更加明显(P0.01)。综上比较说明肝豆扶木汤具有良好的抗肝纤维化临床疗效。(2)实验部分:各组受试动物经治疗2、4、6、8周后的检验指标比较结果如下:⑴模型组2、4、6、8周与对应的正常组比较,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对值显著升高,Smad7m RNA相对值显著降低(P0.01)。⑵各治疗组与对应模型组比较如下:(1)高剂量组、中剂量组、丹参组、青霉胺组2、4、6、8周及低剂量组4、6、8周较对应模型组,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对值均下降,Smad7m RNA相对值上升(P0.05,P0.01);(2)低剂量组2周较对应模型组无统计学差异(P0.05);⑶GDFMT高剂量组与对应GDFMT中、低剂量组、丹参组、青霉胺组比较如下:(1)GDFMT中、低剂量组、丹参组、青霉胺组2、4、6、8周及青霉胺4、6、8周与对应的GDFMT高剂量组比较,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对表达量降低,Smad7m RNA的相对表达量升高(P0.05,P0.01);(2)与青霉胺组2周比较,GDFMT高剂量组与其无统计学差异(P0.05)。⑷GDFMT中剂量组与丹参组、青霉胺组、低剂量组比较如下:(1)中剂量组与丹参组比较无统计学差异(P0.05);(2)与青霉胺组比较,中剂量组2、4周的TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对表达量较其高,Smad7m RNA的相对表达量较其低(P0.05,P0.01);青霉胺组与中剂量组6、8周比较,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对表达量降低,Smad7m RNA的相对表达量升高(P0.05,P0.01);(3)低剂量与对应中剂量组6、8周比较,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对表达量较其低,Smad7m RNA的相对表达量较其高(P0.01);低剂量组2周与对应中剂量比较无差异(P0.05)。⑸丹参组与青霉胺组、低剂量组比较如下:(1)丹参组2、4周较对应青霉胺组TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对表达量较其高,Smad7m RNA的相对表达量较其低(P0.05,P0.01);丹参组6、8周较对应青霉胺组TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对表达量降低,Smad7m RNA的相对表达量升高(P0.05,P0.01);(2)丹参组6、8周与对应低剂量组比较,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对表达量较其低,Smad7m RNA的相对表达量较其高(P0.01);丹参组2周与对应低剂量组比较无差异(P0.05)。⑹青霉胺组与低剂量组比较如下:青霉胺2、4周较对应低剂量组,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对表达量较其低,Smad7m RNA的相对表达量较其高(P0.05,P0.01);青霉胺组6周较对应低剂量组无差异(P0.05);青霉胺8周较对应低剂量组,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4的m RNA相对表达量较高,Smad7m RNA的相对表达量较低(P0.01)。⑺(1)GDFMT高剂量组4周与对应2周相比,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4m RNA相对值降低,Smad7m RNA相对值升高(P0.01);(2)GDFMT中、低剂量组4周与对应2周相比,无明显差异(P0.05)。⑻6周与对应4周比较,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smad4m RNA相对值降低,Smad7m RNA相对值升高(P0.01);⑼8周与对应6周比较,TGF-β1、TβRI、TβRII、Smad2、Smad3和Smadm RNA相对表达量均降低,Smad7m RNA相对表达量则升高(P0.01)。结论:(1)GDFMT能够有效地改善WD肝纤维化患者中医证候积分及UWDRS肝脏功能评分,改善其临床症状和体征。(2)GDFMT能够有效地降低WD肝纤维化患者TGF-β1、TNF-α、IL-6,发挥抗纤维化的作用。(3)GDFMT使TGF-β1、TβRⅠ、TβRⅡ、Smad2、Smad3、Smad4的m RNA相对表达量下降、Smad7m RNA相对表达量升高,说明GDFMT能够调控TGF-β1/Smad信号转导通路中的蛋白表达,抑制TGF-β1/Smad信号传导通路激活HSC,从而发挥抗纤维化作用,且对TGF-β1/Smad信号传导的调控呈量效-时效依赖性。
[Abstract]:Objective: (1) To observe the effect of Gandou Fumu Decoction (GDFMT) on UWDRS liver function score, TCM syndrome score, TGF-beta 1, TNF-a and IL-6 in patients with Wilson's disease (WD) and liver fibrosis treated with GDFMT. (2) Based on the TGF-beta 1/smad signal transduction pathway and TX mice, Gandou Fumu Decoction (GDFMT) was used as an intervention drug to treat WD hepatic fibrosis. After the intervention of different courses of high, medium and low concentration GDFMT, the liver tissues were detected by semi-quantitative RT-PCR. TGF-beta 1, Tbeta RI, Tbeta RII, Smad 2, Smad 3, Smad 4 and Smad 7 were used to investigate the effect of GDFMT on hepatic fibrosis induced by WD and its mechanism. Shen granules were given the same basic treatment as those in the two groups. The basic treatment was 8 days as a course of treatment. DMPS was given intravenous copper-repelling therapy for 6 days, and calcium-supplementing therapy was given 2 days after treatment for 4 courses. In the experimental part, 192 male TX mice were randomly divided into 6 groups: high dose GDFMT group, middle dose GDFMT group, low dose GDFMT group (abbreviated as "high dose group, middle dose group, low dose group"), model group (saline), penicillamine group (penicillamine), Salvia miltiorrhiza group (Salvia miltiorrhiza), and 32 DL mice as normal control group (7 groups). Seven groups of animals were given corresponding drug treatment once a day for 56 consecutive days. At the end of 2,4,6,8 weeks after the treatment, the animals were sacrificed in batches, 8 in each group. The relative expression of TGF-beta 1, Tbeta RI, Tbeta RII, Smad2, Smad3, Smad4 and S7 in liver tissues of TX mice was detected by semi-quantitative RT-PCR. Results: (1) Clinical part: After four courses of treatment, the total clinical efficacy of the treatment group was significantly better than that of the control group (P After treatment, the scores of TCM syndromes decreased (P 0.01, P 0.05); compared with the same group before treatment, the scores of UWDRS liver function in the two groups after treatment were lower, with statistical significance (P 0.01, P 0.05); after treatment, the scores of TCM syndromes in the two groups were compared, the treatment group was more significantly lower than the control group (P 0.01); after treatment, the scores of UWDRS liver function in the two groups were significantly lower than those in the control group (P 0.01); The therapeutic effect of the treatment group was better than that of the control group (P 0.01). Evaluation of clinical therapeutic indexes: serum TGF-beta 1, TNF-a, IL-6 before treatment were compared between the treatment group and the control group, there was no difference in TGF-beta 1, TNF-a, IL-6 before and after treatment between the two groups (P 0.05); before and after treatment, the treatment group compared with the pre-treatment group, the treatment group TGF-beta 1, TNF-a, IL-6 were significantly reduced (P 0.01). After treatment, TGF-beta 1, TNF-alpha and IL-6 in the control group decreased significantly (P 0.01, P 0.05); after treatment, compared with the control group, the treatment group improved more significantly (P 0.01). To sum up, Gandou Fumu Decoction has a good clinical effect on anti-hepatic fibrosis. (2) Experimental part: The animals in each group were tested after 2, 4, 6, 8 weeks of treatment. The results were as follows: (1) Compared with the corresponding normal group, the relative value of M RNA of TGF-beta 1, Tbeta RI, Tbeta RII, Smad2, Smad3 and Smad 4 in the model group increased significantly at 2, 4, 6 and 8 weeks, and the relative value of Smad7m RNA decreased significantly (P 0.01). (2) The comparison between the treatment groups and the corresponding model group was as follows: (1) High dose group, middle dose group, Salvia miltiorrhiza group, Penicillamine group at 2, 4, 4, 6, 8 weeks and low. Compared with the corresponding model group, the relative value of M RNA of TGF-beta 1, Tbeta RI, Tbeta RII, Smad2, Smad3 and Smad 4 decreased, while the relative value of Smad7m RNA increased (P Compared with the corresponding GDFMT high-dose group, the relative expression of M RNA of TGF-beta 1, T-beta RI, T-beta RII, Smad2, Smad3 and Smad 4 decreased, while the relative expression of Smad7m RNA increased (P 0.05, P 0.01) in the middle, low-dose group, the salvia miltiorrhiza group, the penicillamine group and the penicillamine group at 2, 4, 6, 8 weeks; (2) Compared with the penicillamine group at 2 weeks, there was no significant difference between the high-dose group and the corresponding GDFMT high-dose group (P 0.05). (2) Compared with the Penicillin group, the relative expression of TGF-beta 1, Tbeta RI, Tbeta RII, Smad2, Smad3 and Smad 4 in the middle dose group was higher, and the relative expression of Smad7m RNA was lower (P 0.05, P 0.0). 1) Compared with the medium-dose group, the relative expression of M RNA of TGF-beta 1, Tbeta RI, Tbeta RII, Smad2, Smad 3 and Smad4 decreased, while the relative expression of Smad7m RNA increased (P 0.05, P 0.01); (3) Compared with the corresponding medium-dose group at 6 and 8 weeks, the relative expression of TGF-beta 1, Tbeta RI, Tbeta RII, Smad2, Smad 3 and Smad4 was lower, and the relative expression of Smad7m RNA was lower. The expression of TGF-beta 1, T-beta RI, T-beta RII, Smad2, Smad3 and Smad 4 in the salvia miltiorrhiza group was higher than that in the corresponding penicillamine group at 2 and 4 weeks, and the relative expression of Smad7m RNA was lower than that in the corresponding penicillamine group (P 0.05, P 0.01). Compared with the corresponding penicillamine group, the relative expression of TGF-beta 1, T-beta RI, T-beta RII, Smad2, Smad3 and Smad4 in Danshen group decreased at 6 and 8 weeks, while the relative expression of Smad7m RNA increased (P 0.05, P 0.01); (2) Compared with the corresponding low-dose group at 6 and 8 weeks, the relative expression of TGF-beta 1, T-beta RI, T-beta RII, Smad2, Smad3 and Smad4 was lower, and the relative expression of Smad7m RNA was lower in Danshen group. Compared with the corresponding low-dose group, the relative expression of M RNA of TGF-beta 1, T-beta RI, T-beta RII, Smad2, Smad3 and Smad 4 in the penicillamine group was lower, and the relative expression of Smad7m RNA was higher (P 0.05, P 0.01). Compared with the corresponding low-dose group, there was no significant difference (P 0.05); the relative expression of M RNA of TGF-beta 1, T-beta RI, T-beta RII, Smad2, Smad3 and Smad 4 was higher in the penicillamine group at 8 weeks, and Smad7m RNA was lower in the high-dose group (P 0.01). Relative RNA value increased (P 0.01); (2) In GDFMT, there was no significant difference between the low-dose group and the corresponding two weeks (P 0.05). _Compared with the corresponding four weeks, the relative values of TGF-1, T-beta RI, T-beta RII, Smad2, Smad3 and Smad4m RNA decreased, and the relative value of Smad7m RNA increased (P 0.01); Compared with the corresponding six weeks, the relative tables of TGF-beta 1, T-beta RI, T-beta RI, Smad2, Smad3 and Smad4m RNA increased (P 0.01); _Compared with the corresponding six weeks, the relative tables of TGF Conclusion: (1) GDFMT can effectively improve TCM syndrome score and UWDRS liver function score in patients with WD liver fibrosis, and improve their clinical symptoms and signs. (2) GDFMT can effectively reduce TGF-beta 1, TNF-a, IL-6 in patients with WD liver fibrosis, and play an anti-fibrosis role. (3) GDFMT can make T. GF-beta-1, T-beta-R I, T-beta-R II, Smad2, Smad3, Smad4 relative expression of M RNA decreased, Smad7m RNA relative expression increased, indicating that GDFMT can regulate the expression of protein in the TGF-beta 1/Smad signal transduction pathway, inhibit the TGF-beta 1/Smad signal transduction pathway to activate HSC, thereby playing an anti-fibrosis role, and has a dose-effect on the regulation of TGF-beta 1/Smad signal transduction. Time dependence.
【学位授予单位】：安徽中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R742.4

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