平突散系列复方对CD40L刺激后人球后成纤维细胞的MMP和TIMP表达的影响

Effects of Pingtusan Compounds on the Expression of MMP and TIMP in Retroocular Fibroblasts after CD40L Stimulation

背景 尽管目前关于甲状腺相关性眼病(TAO)在基础和临床研究方面已取得了一些进展,同时积累了一定量的关于发病机制的材料和证据,但目前国内外关于TAO的研究仍然很少,临床仍缺少有效的治疗方法和药物.与此同时,在传统中医领域,中医药辨证治疗TAO疗效肯定、不良反应少且复发率低,但其作用机制不明确,药物作用靶点不明.目的 观察CD40L对人球后成纤维细胞(RFs)表面基质金属蛋白酶(MMP)-1、MMP-2、MMP组织抑制剂(TIMP)-1、TIMP-2表达的影响以及平突散系列中药复方的干预效果.方法 2016年9月选取35只SPF级SD雄性大鼠用于制备泻火平突散、活血平突散、养目平突散含药血清.实验中所用的人眼球后结缔组织来源于2例眼创伤患者(2014年取材于郑州大学第一附属医院眼科),体外培养RFs成功后,随机分为7组,其中空白组:给予正常培养液;血清组:给予含10% 正常大鼠血清的培养液;CD40L组:给予含100 ng/ml CD40L的培养液;CD40L+血清组的培养液:给予含100 ng/ml CD40L和10% 正常大鼠血清的培养液;泻火平突散组:给予含有100 ng/ml CD40L和10% 含药血清的培养液;活血平突散组:给予含有100 ng/ml CD40L与10% 含药血清的培养液;养目平突散组:给予含有100 ng/ml CD40L与10% 含药血清的培养液.免疫细胞化学染色法检测各组MMP-1、MMP-2、TIMP-1、TIMP-2的表达.结果 CD40L组MMP-1、MMP-2活性低于空白组,CD40L+血清组MMP-1、MMP-2活性低于血清组(P<0.05);泻火平突散、活血平突散、养目平突散组MMP-1、MMP-2活性高于CD40L+血清组(P<0.05);养目平突散组MMP-1、MMP-2活性高于泻火平突散组、活血平突散组(P<0.05).CD40L组TIMP-1、TIMP-2活性高于空白组,CD40L+血清组TIMP-1、TIMP-2活性高于血清组(P<0.05);泻火平突散、活血平突散、养目平突散组TIMP-1、TIMP-2活性低于CD40L+血清组(P<0.05);养目平突散组TIMP-1、TIMP-2活性低于泻火平突散组、活血平突散组(P<0.05).CD40L组MMP-1/TIMP-1、MMP-2/TIMP-2低于空白组,CD40L+血清组MMP-1/TIMP-1、MMP-2/TIMP-2低于血清组(P<0.05);泻火平突散、活血平突散、养目平突散组MMP-1/TIMP-1、MMP-2/TIMP-2高于CD40L+血清组(P<0.05);养目平突散组MMP-1/TIMP-1、MMP-2/TIMP-2高于泻火平突散组、活血平突散组(P<0.05).结论 平突散系列复方可能通过阻断CD40-CD40L共刺激信号通路导致MMP/TIMP的失衡,而达到治疗TAO的目的.

Background Regarding thyroid-associated ophthalmopathy(TAO),although modern medicine has made some progress in basic and clinical research,and accumulated a certain amount of materials and evidence on pathogenesis, there are still few studies on TAO treatment,and there are still a lack of effective therapeutic methods and drugs in clinical practice.Furthermore,for TAO,TCM-based syndrome differentiation and treatment has a certain therapeutic effect with few side effects and low recurrence rate,but the mechanism of action is not clear,and the target of drug action is unclear.Objective To observe the effects of CD4 0L on the expression of MMP-1,MMP- 2,TIMP- 1,TIMP- 2 on the surface of retroocular fibroblasts (RFs) and the intervention results of three TCM composite drugs.Methods In this experiment,the serum contained in three TCM composite drugs(xiehuopingtusan,huoxuepingtusan and yangmupingtusan) was derived from 35 male SPF SD rats in September 2016,and the human retrobulbar connective tissue was derived from two patients with ocular trauma(from Department of Ophthalmology,the First Affiliated Hospital of Zhengzhou University in 2014).Successfully cultured RFs were randomly divided into 7 groups:blank group(cultured in a normal medium),serum group(cultured in a medium containing 10% normal rat serum),CD40L group(cultured in a culture medium containing 100 ng/ml CD40L),CD40L+serum group (cultured in a medium containing 100 ng/ml CD40L and 10% normal rat serum),xiehuopingtusan group(cultured in a medium containing 100 ng/ml CD4 0L and 10% xiehuopingtusan-containing rat serum with),huoxuepingtusan group(cultured in a medium containing 100 ng/ml CD40L and 1 0% huoxuepingtusan-containing rat serum), and yangmupingtusan group(cultured in a medium containing 100 ng/ml CD4 0L and 10% yangmupingtusan-containing serum).Immunocytochemical staining was used to detect the expression of MMP-1,MMP- 2,TIMP- 1 and TIMP-2 in each group.Results The activities of MMP-1 and MMP- 2 in CD4 0L group were lower than those of the blank group(P<0. 05).T he activities of MMP-1 and MMP-2 in CD4 0L+serum group were lower than those of serum group,and of the three TCM composite drug groups(P<0. 05).T he activities of MMP-1 and MMP-2 in yangmupingtusan group were higher than those in xiehuopingtusan group and huoxuepingtusan group(P<0. 05). The activities of TIMP-1 and TIMP-2 in CD4 0L group were higher than those in blank group(P<0. 05).T he activities of TIMP- 1 and TIMP-2 in CD40L+serum group were higher than those in serum group,and those in three TCM composite drug groups (P<0. 05).The activities of TIMP-1 and TIMP-2 in yangmupingtusan group were lower than those in xiehuopingtusan group and huoxuepingtusan group(P<0.05).Mean ratios of MMP-1/TIMP-1 and MMP-2/TIMP-2 in the CD40L group were lower than those in the blank group(P<0. 05).Mean ratios of MMP-1/TIMP- 1 and MMP-2/TIMP- 2 in CD40L+serum group were lower than those in serum group(P<0. 05).Mean ratios of MMP-1/TIMP- 1 and MMP-2/TIMP- 2 in three TCM composite drug groups were higher than those in CD4 0L+serum group(P<0. 05).Mean ratios of MMP-1/TIMP- 1 and MMP-2/TIMP- 2 in yangmupingtusan group were higher than those in the xiehuopingtusan group and huoxuepingtusan group(P<0. 05). Conclusion The treatment of TAO by TCM composite drugs may be achieved by blocking the CD4 0-CD 40L co-stimulatory pathway,subsequently leading to the imbalance of MMP/TIMP.