注射用维通对腹部术后粘连的药效评价与作用机制研究
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摘要
研究背景
腹部粘连是腹部手术后的常见并发症,主要表现为原本分离的脏器之间或脏器与腹壁之间出现范围不定的粘连或形成纤维索带,可导致疼痛、肠梗阻、女性不孕等病症。虽然腹腔粘连引起的症状可通过粘连松解术缓解,但复发几率仍然很大,高达55%～95%。近年来,随着中医药研究的飞速发展,很多中药经方和验方被应用到临床治疗中。中药以其多靶点发挥疗效、毒副作用小、易被患者接受等优势,成为防治术后粘连又一重要途径,具有很大的研究前景与开发价值。
注射用维通(Weitong Injection,WTI)是南方医院药学部在常通口服液(Changtong Oral Liquid,CTOL)研究基础上,研制开发的纯中药粉针剂型。含有改善微循环、抑制炎症反应、抗渗出的活性成分和药理功效,临床主要用于防治术后粘连。该药具有起效快和不受术后禁食期限制等优点,与常通口服液配套使用,腹部手术后早期使用静脉滴注制剂(WTI),恢复饮食以及出院后使用口服制剂(CTOL),是术后粘连防治体系中的又一重要新药。
目的
通过在体的动物药效学实验与离体的细胞生物学、分子生物学实验,结合现代化学分析技术,分别从机体的整体水平与细胞分子水平探讨WTI防治术后粘连的作用机制,确定其主要活性成分,为该药的临床应用提供理论依据与数据支持。
方法
1 WTI对大鼠术后粘连模型的作用研究
大鼠分为正常对照组、模型对照组、地塞米松组、WTI高、低剂量组,除正常对照组外均采用多因素法制备成术后粘连模型,其中WTI高、低剂量组从手术当日起,每日分别按2.4、1.2mg·kg~(-1)尾静脉注射WTI,连续7d;地塞米松组按每日5mg·kg~(-1)尾静脉注射地塞米松注射液,模型对照组与正常对照组每日静脉注射生理盐水1mL·kg~(-1),均连续7d。各组于术后7d给药1hr后麻醉开腹,评价腹腔组织粘连程度,腹腔静脉采血检查血常规,取正常与粘连的盲肠壁组织作病理切片检查,并检测羟脯氨酸(Hydroxyproline,HyP)含量。
2 WTI主要成分的分析研究
2.1 WTI中D含量测定
色谱条件为:流动相:磷酸盐缓冲液(0.5%NaH_2PO_4-0.03%H_3PO_4):甲醇=95:5;检测波长220nm;流速:1mL·min~(-1);色谱柱:Luna~(?) 5u C18(2)100A色谱柱(150×4.60mm,5μm);柱温:室温。在此条件下进行线性范围考察、阴性对照试验、精密度实试验、回收率试验、稳定性试验与WTI样品测定。
2.2 WTI中Q含量测定
流动相:乙腈:1%磷酸溶液=40:60;检测波长220nm;流速:1mL·min~(-1);色谱柱:Luna~(?) 5u C18(2)100A色谱柱(150×4.60mm,5μm,美国Phenomenex公司);柱温:室温。在此条件下进行线性范围考察、阴性对照试验、精密度实试验、回收率试验、稳定性试验与WTI样品测定。
3 WTI对正常腹膜与粘连腹膜成纤维细胞(FB)增殖的作用研究
3.1 WTI对正常腹膜与粘连腹膜成纤维细胞增殖活性的作用
取大鼠制备成术后粘连模型,术后7d分别取正常与粘连腹壁组织作FB原代培养,培养条件为:含10%FBS的DMEM培养基,37℃,5%CO_2,95%湿度。传代后取3～5代FB进行实验。将NFB与AFB分别接种至96孔板上,均设WTI、D对照品、Q对照品、D+Q对照品、地塞米松注射剂5个组,每组均设6个药物处理浓度,分别为0、2.5、5.0、10.0、20.0、40.0μg·mL~(-1),其中D+Q对照品组的药液按照WTI中D与Q的含量比例配制(D:Q=1:10),以相同的条件培养72hr,MTT法测定各孔吸光度,计算出各个浓度下的生长抑制率(IR),并求出各药的半数抑制率(IC_(50))。
3.2 WTI对正常腹膜与粘连腹膜成纤维细胞细胞周期的作用
取与3.1相同的NFB与AFB接种至培养皿中,均设空白、WTI、D对照品、Q对照品、D+Q对照品、地塞米松注射剂6个组,其中D+Q对照品组的药液按照10.0μg·mL~(-1)WTI中D与Q的含量比例配制(D:Q=1:10),其余药物处理组在相同培养条件下以10.0μg·mL~(-1)的药物浓度培养72hr,流式细胞术(FCM)检测各组FB的细胞周期。
3.3 WTI对正常腹膜与粘连腹膜成纤维细胞细胞超微结构的作用
取与3.1相同的NFB与AFB接种至培养皿中,设空白组与WTI组,其中WTI组的药物浓度为10μg·mL~(-1),在相同的条件下培养72hr,收集各组细胞,经固定、切片、染色等处理后,在透射电镜下观察其超微结构。
4 WTI对正常腹膜与粘连腹膜成纤维细胞目的基因mRNA表达与合成产物的作用研究
取与3.1相同的NFB与AFB,提取总RNA,利用内参照β-actin的引物与目的基因TGF-β_1、TGF-β_2、MMP-1、TIMP-1的引物进行RT-PCR。扩增产物经琼脂糖凝胶电泳分离后进行纯化,得到定量标准模板,按倍比稀释得到不同浓度的标准模板,加入相应的荧光探针与引物,在Real-time PCR仪上进行PCR扩增,将不同浓度的标准模板拷贝的对数和相应的C_T值(Threshold Cycle)作图,得到内参照与各目的基因的标准曲线。
取与3.1相同的NFB与AFB接种至培养皿中,均设空白、WTI、D对照品、Q对照品、D+Q对照品、地塞米松注射剂6个组,其中D+Q对照品组的药液按照10.0μg·mL~(-1)WTI中D与Q的含量比例配制(D:Q=1:10),其余药物处理组在相同培养条件下以10.0μg·mL~(-1)的药物浓度培养72hr,提取各组细胞的总RNA,逆转录为cDNA后,加入相应的荧光探针与引物,在Real-time PCR仪上进行PCR扩增,测定各组的C_T值,从相应目的基因标准曲线上求出各自的起始模板拷贝数,与内参照β-actin的拷贝数相比,求出各组FB不同目的基因(TGF-β_1、TGF-β_2、MMP-1、TIMP-1)的相对表达量。同时收集NFB与AFB各组培养72hr后的培养液,碱水解比色法测定HyP浓度,测定胶原合成水平。
结果
1 WTI对大鼠术后粘连模型的作用研究
1.1 WTI对大鼠粘连程度的影响
大鼠各实验组粘连程度均存在显著性差异(P＜0.05)。大鼠WTI高、低剂量组与地塞米松的平均秩次组均低于模型对照组,WTI高剂量组与地塞米松组的平均秩次也低于WTI低剂量组;
1.2 WTI对大鼠血液指标的影响
模型对照组WBC与其余各组有显著性差异(P＜0.05),各给药组WBC与正常对照组无显著性差异,各给药组之间WBC也无显著性差异(P＞0.05);WTI高剂量组RBC与地塞米松组有显著性差异(P＜0.05);各组HGB、LYM无显著性差异(P＞0.05);模型对照组PLT与WTI低剂量组无显著差异(p＞0.05),但与其余各组有显著性差异(P＜0.05),地塞米松组与正常对照组、WTI高剂量组均无显著差异(P＞0.05);模型对照组NEU与其余各组存在显著性差异(P＜0.05),其余各组间NEU无显著性差异(P＞0.05)。
1.3 WTI对大鼠盲肠壁组织HyP含量的影响
正常对照组HyP低于其余各组,并有显著性差异(P＜0.05);模型对照组HyP高于其余各组,也有显著性差异(P＜0.05);地塞米松组与WTI低剂量组无显著性差异(P＞0.05),与WTI高剂量组有显著性差异(P＜0.05)。
1.4大鼠病理切片观察
正常盲肠壁结构基本清晰,可见粘膜、粘膜下层、肌层、浆膜层四层结构。粘膜固有层内可见少量炎性细胞浸润,粘膜下层可见少量疏松结缔组织,肌层内环形及外纵层平滑肌结构清晰。浆膜层很薄,外部覆盖间皮细胞;模型对照组可见盲肠组织与结肠、小肠粘连,粘连带中有较多纤维组织增生并伴有充血及大量炎性细胞浸润,纤维组织甚至可见玻璃样变;WTI低剂量组可见盲肠组织中轻度粘连,纤维组织增生并伴有充血及炎性细胞浸润;WTI高剂量组可见部分盲肠组织中少量纤维组织增生并伴有散在炎性细胞浸润;地塞米松组与WTI高剂量组情况相似。
2 WTI主要成分的分析研究
测得WTI中D含量为4.99%,Q-A含量为14.98%,Q-B含量为11.42%,采用的色谱方法不受其它组分的干扰,精密度、回收率、稳定性良好,线性范围可满足测定的需要。
3 WTI对正常腹膜与粘连腹膜成纤维细胞增殖的作用研究
3.1 WTI对FB增殖活性的影响
AFB各药物处理组的IC_(50)均与NFB存在显著性差异(P＜0.05),WTI及其主要成分、地塞米松对AFB的IC_(50)值低于NFB。剂量-光密度曲线显示:AFB的WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的OD值下降幅度高于NFB各组。
3.2 WTI对FB细胞周期的影响
AFB空白组G_0/G_1期与NFB空白组无显著性差异(P＞0.05),G_2/M期与NFB空白组有显著差异(P＜0.05),其中S期细胞百分数高于NFB空白组,G_2/M期细胞百分数低于NFB空白组;AFB各药物处理组除地塞米松组外,G_0/G_1期、S期、G_2/M期均与NFB相应的药物处理组有显著性差异(P＜0.05),G_0/G_1期细胞百分数均高于NFB组,S期、G_2/M期细胞百分数均低于NFB组;AFB地塞米松组G_0/G_1期、G_2/M期与NFB地塞米松组有显著性差异(P＜0.05),S期与NFB地塞米松组无显著性差异(P＞0.05),其中G_0/G_1期细胞百分数高于NFB地塞米松组,G_2/M期细胞百分数低于NFB地塞米松组。
3.3 WTI对FB超微结构的影响
观察比较NFB与AFB未加药培养时的超微结构,未见有明显差异。当加入WTI培养后,AFB溶酶体数目增多,脂滴数目较多,内质网有扩张现象,线粒体有空泡变,嵴不明显。而NFB脂滴数目较少,溶酶体数目少,内质网正常,有明显的高尔基体存在。由此说明WTI可对NFB与AFB的超微结构产生影响,这应与FB增殖活性受到抑制有关。
4 WTI对正常腹膜与粘连腹膜成纤维细胞目的基因mRNA表达与合成产物的作用研究
4.1 TGF-β_1的相对表达量
AFB与NFB空白组的TGF-β_1 mRNA相对表达量有显著差异(P＜0.05);NFB的各个药物处理组均与空白组存在显著差异(P＜0.05);与空白组相比,WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的TGF-β_1mRNA相对表达量分别下降14.94%、8.11%、9.86%、10.33%、8.11%;AFB的各个药物处理组均与空白组存在显著差异(P＜0.05),与空白组相比,WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的TGF-β_1 mRNA相对表达量分别下降19.07%、26.69%、26.91%、16.14%、8.05%。
4.2 TGF-β_2的相对表达量
AFB与NFB空白组的TGF-β_2 mRNA相对表达量有显著差异(P＜0.05);NFB的各个药物处理组均与空白组存在显著差异(P＜0.05),与空白组相比,WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的TGF-β_1mRNA相对表达量分别下降17.02%、18.92%、33.45%、18.56%、8.98%;AFB的各个药物处理组均与空白组存在显著差异(P＜0.05),与空白组相比,WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的TGF-β_2 mRNA相对表达量分别下降40.47%、42.97%、49.24%、40.94%、20.41%。
4.3 MMP-1与TIMP-1的相对表达量
AFB与NFB空白组的MMP-1、TIMP-1 mRNA相对表达量均有显著差异(P＜0.05);NFB除地塞米松组的MMP-1与空白组无显著差异外(P＞0.05),其余各药物处理组均与空白组存在显著差异(P＜0.05);AFB的各个药物处理组均与空白组存在显著差异(P＜0.05)。
AFB与NFB空白组的MMP-1/TIMP-1比值存在显著性差异(P＜0.05),AFB的MMP-1/TIMP-1比值高于NFB;AFB各个药物处理组与AFB空白组均存在显著差异(P＜0.05),WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的MMP-1/TIMP-1比值分别增加14.74%、17.31%、21.79%、21.77%、11.54%;NFB的各个药物处理组与NFB空白组均无显著差异(P＞0.05)。
4.4胶原合成水平
AFB与NFB空白组的HyP存在显著性差异(P＜0.05),AFB的HyP高于NFB;AFB各个药物处理组与AFB空白组均存在显著差异(P＜0.05),WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的HyP含量分别降低43.05%、39.68%、37.75%、36.75%、46.43%;NFB各个药物处理组与NFB空白组均无显著差异(P＞0.05)。
结论
1 WTI可减少腹膜受损处的胶原过度合成,降低该部位的纤维化程度,同时可减少炎症反应,降低过高的血液粘度,从而发挥粘连防治作用。
2 WTI的主要活性成分为D与Q。
3 WTI及其主要成分对FB增殖均有抑制作用,而且对AFB的作用比NFB强;同时还可调整FB的细胞周期,使其受阻于G_1期,延缓向S期、G2期过渡的速度,进而阻止FB进入M期,达到抑制其增殖的效果,对AFB调节作用也强于NFB。
4 WTI及其主要成分能有效降低AFB的TGF-β_1、TGF-β_2 mRNA表达水平,降低AFB的增殖速度,同时通过调节AFB的MMP-1与TIMP-1相对表达量比例,加快ECM降解速率,减少胶原合成量,从而减少粘连部位的纤维化程度。
Background
Post-surgical adhesion is a quite common complication,especially after general laparotomy.It manifests with fibrous bands joining normally separated organs together with the intestinal/abdominal wall.Intra-abdominal adhesion formation and re-formation after surgery is a significant cause of morbidity,such as pain,bowel obstruction,even infertility.According to previous studies,although recent cases suggests that a proportion of people suffering such adhesion may benefit from adhesiolysis,adhesion re-formation still remains as the most serious post-surgical problem,the probability ranging from 55%to 95%among the patients even those who have had undergone surgical adhesion removal.Up to now,a number of TCM (Traditional Chinese Medicine) treatment and herbal preparation have been evaluated for their value in post-surgical adhesion prevention.So it is necessary to exert the superiority and characteristic of TCM in treating post-surgical adhesion.
WTI(Weitong Injection),developed by Department of pharmaceutics,Nanfang Hospital,is a pure herbal injectable powder for anti-adhesion.It bases on the study of Changtong Oral Liqud and contains components from herb,which can improve microcirculation of blood,decrease inflammatory reaction and exudation.It is another important new drug for post-surgical adhesion.
Objective
To evaluate and explore the pharmacodynamic action and mechanism of WTI with the following experiments:To observe the effect of WTI on post-surgical adhesions by establishing rat experimental models with intestinal adhesions.To detect the main components of WTI by HPLC.To explore the effect of WTI and its components on proliferation and cell cycle of cultured fibroblasts from normal and adhesive peritoneal tissues.To detect synthesis product and mRNA expression of cytokines from NFB and AFB.
Methods
1.Pharmacodynamic action of WTI
Rats were received laparotomy and prepared as intestinal adhesion model.WTI was intravenously injected for 7 days.Dexamethasone were used as positive control. Blood routine examination was conducted.Intraperitoneal adhesions were graded by adhesion score on day 7.Tissues from normal and adhesive tissues were collected to prepare pathological sections.
2.Detection of components in WTI
Components of WTI were determined by HPLC.The mobile phase of D was phosphate buffer solution:methanol(95:5).The mobile phase of Q was 1% phosphoric acid:acetonitrile(60:40).Flow rate was 1mL·min~(-1).The UV detective wavelength was 220nm.Chromatographic column was Luna~(?) 5u C18(2) 100A. Column temperature was room temperature.
3.Effect of WTI on proliferation and cell cycle of NFB and AFB
The normal and adhesive peritoneal tissues were harvested from adhesion model of rots.The prepared tissues were cultured for 1～2 weeks until outgrowth of NFB and AFB.The cells of 3～5 passage were seeded on 96-well plates at a density of approximately 5×10~3 cells per well in 10%FBS.Then each kind of FB was divided into 5 groups:WTI,D standard,Q standard,D+Q standard and dexamethasone group. Each group had 6 different drug concentration ranging from 0～40μg·mL~(-1).The dosage of D+Q standard group was based on the ratio of D and Q in WTI(D:Q=1:10). Cultured for 72hr,the proliferation of NFB and AFB were detected by MTT assay.
The cells of 3～5 passage were seeded on culture dishes at a density of approximately 1×10~5 cells per dish in 10%FBS.Then each kind of FB was divided into 6 groups:blank,WTI,D standard,Q standard,D+Q standard and dexamethasone group.The dosage of D+Q standard group was based on the ratio of D and Q in 10μg·mL~(-1)WTI(D:Q=1:10).The drug concentration of other drug groups was 10μg·mL~(-1).Cultured for 72hr,the cell cycle of NFB and AFB were analyzed by FCM.
The cells of 3～5 passage were seeded on culture dishes at a density of approximately 1×10~5 cells per dish in 10%FBS.Then each kind of FB was divided into 2 groups:blank and WTI group.The drug concentration of WTI group is 10μg·mL~(-1).Cultured for 72hr,then cells were collected to observe the ultramicrostructure by transmission electron microscope.
4.Effect of WTI on cytokines mRNA expression and collagen synthesis of NFB and AFB
The cells of 3～5 passage were seeded on culture dishes at a density of approximately 1×10~5 cells per dish in 10%FBS.Then each kind of FB was divided into 5 groups:blank,WTI,D standard,Q standard,D+Q standard and dexamethasone group.The dosage of D+Q standard group was based on the ratio of D and Q in 10μg·mL~(-1)WTI(D:Q=1:10).The drug concentration of other drug groups was 10μg·mL~(-1).Cultured for 72hr,cells were collected to extract total RNA.Then the RT reaction was carried out on each RNA sample to produce cDNA.Real-time PCR was performed in a reaction plate containing RT products(cDNA),TaqMan fluorescent probe,relative primer and other PCR reagents.C_T of each sample was recorded. According to the standard curve ofβ-actin and each target gene,the copy number of original templates(β-actin,TGF-β_1,TGF-β_2,MMP-1,TIMP-1)were obtained.Finally, the absolute copy number of target gene was normalized to that ofβ-actin.On the other hand,collected the supernatant of cells and determine HyP by base hydrolysis-chromatometry.
Results
1.WTI could significantly reduce the level of adhesion,lower WBC and NEU counting,recover PLT counting to the preliminary level.WTI could also lower HyP concentration in traumatized tissues.Pathological sections showed the inflammation level of WTI groups were much lower than model group.Peritoneal and cecal wall of WTI groups were also fixed.Such effect of WTI were better than dexamethasone.
2.D and Q were the main components of WTI.The methods built up for HPLC were fit for the assaying of these components.The content of D was 4.99%.The content of Q-A and Q-B were 14.98%and 11.42%respectively.
3.IC_(50) of all AFB groups were lower than NFB groups.Dexamethosone groups had the highest IC_(50) among all AFB groups.Compared with blank groups,all drug groups had higher G_0/G_1 proportion and lower S,G_2/M proportion.Compared with all drug groups of NFB,all drug groups of AFB had higher G_0/G_1 proportion and lower S, G_2/M proportion.Transmission electron microscope picture showed WTI could change the ultramicrostructure of AFB.
4.Compared with blank group,the mRNA level of TGF-β_1,TGF-β_2,MMP-1, TIMP-1 of all drug groups were lower.Among the AFB groups,MMP-1/TIMP-1 mRNA ratio of drug groups were higher than blank group,and there was no significant difference between NFB drug groups and NFB blank group.Compared with blank groups of AFB,drug groups of AFB had lower HyP in the supernatant,and there was no significant difference between NFB drug groups and NFB blank group.
Conclusions
1.WTI could reduce excess synthesis of collagen in traumatized peritoneum and decrease fibrosis level.WTI could also reduce inflammatory reaction and improve the high blood viscosity caused by trauma.
2.D and Q were the main components of WTI.
3.Both WTI and its main components could regulate the cell cycle of FB and restrain the proliferation of FB.And AFB was more sensitive to medication than NFB.
4.Both WTI and its main components could lower mRNA expression level of TGF-β_1 and TGF-β_2 in AFB to restrain the proliferation of FB.They could also regulate MMP-1/TIMP-1 mRNA ratio in AFB to decrease synthesis of collagen,which lead to the inhibition of excess fibrosis.
腹部粘连是腹部手术后的常见并发症,主要表现为原本分离的脏器之间或脏器与腹壁之间出现范围不定的粘连或形成纤维索带,可导致疼痛、肠梗阻、女性不孕等病症。虽然腹腔粘连引起的症状可通过粘连松解术缓解,但复发几率仍然很大,高达55%～95%。近年来,随着中医药研究的飞速发展,很多中药经方和验方被应用到临床治疗中。中药以其多靶点发挥疗效、毒副作用小、易被患者接受等优势,成为防治术后粘连又一重要途径,具有很大的研究前景与开发价值。
注射用维通(Weitong Injection,WTI)是南方医院药学部在常通口服液(Changtong Oral Liquid,CTOL)研究基础上,研制开发的纯中药粉针剂型。含有改善微循环、抑制炎症反应、抗渗出的活性成分和药理功效,临床主要用于防治术后粘连。该药具有起效快和不受术后禁食期限制等优点,与常通口服液配套使用,腹部手术后早期使用静脉滴注制剂(WTI),恢复饮食以及出院后使用口服制剂(CTOL),是术后粘连防治体系中的又一重要新药。
目的
通过在体的动物药效学实验与离体的细胞生物学、分子生物学实验,结合现代化学分析技术,分别从机体的整体水平与细胞分子水平探讨WTI防治术后粘连的作用机制,确定其主要活性成分,为该药的临床应用提供理论依据与数据支持。
方法
1 WTI对大鼠术后粘连模型的作用研究
大鼠分为正常对照组、模型对照组、地塞米松组、WTI高、低剂量组,除正常对照组外均采用多因素法制备成术后粘连模型,其中WTI高、低剂量组从手术当日起,每日分别按2.4、1.2mg·kg~(-1)尾静脉注射WTI,连续7d;地塞米松组按每日5mg·kg~(-1)尾静脉注射地塞米松注射液,模型对照组与正常对照组每日静脉注射生理盐水1mL·kg~(-1),均连续7d。各组于术后7d给药1hr后麻醉开腹,评价腹腔组织粘连程度,腹腔静脉采血检查血常规,取正常与粘连的盲肠壁组织作病理切片检查,并检测羟脯氨酸(Hydroxyproline,HyP)含量。
2 WTI主要成分的分析研究
2.1 WTI中D含量测定
色谱条件为:流动相:磷酸盐缓冲液(0.5%NaH_2PO_4-0.03%H_3PO_4):甲醇=95:5;检测波长220nm;流速:1mL·min~(-1);色谱柱:Luna~(?) 5u C18(2)100A色谱柱(150×4.60mm,5μm);柱温:室温。在此条件下进行线性范围考察、阴性对照试验、精密度实试验、回收率试验、稳定性试验与WTI样品测定。
2.2 WTI中Q含量测定
流动相:乙腈:1%磷酸溶液=40:60;检测波长220nm;流速:1mL·min~(-1);色谱柱:Luna~(?) 5u C18(2)100A色谱柱(150×4.60mm,5μm,美国Phenomenex公司);柱温:室温。在此条件下进行线性范围考察、阴性对照试验、精密度实试验、回收率试验、稳定性试验与WTI样品测定。
3 WTI对正常腹膜与粘连腹膜成纤维细胞(FB)增殖的作用研究
3.1 WTI对正常腹膜与粘连腹膜成纤维细胞增殖活性的作用
取大鼠制备成术后粘连模型,术后7d分别取正常与粘连腹壁组织作FB原代培养,培养条件为:含10%FBS的DMEM培养基,37℃,5%CO_2,95%湿度。传代后取3～5代FB进行实验。将NFB与AFB分别接种至96孔板上,均设WTI、D对照品、Q对照品、D+Q对照品、地塞米松注射剂5个组,每组均设6个药物处理浓度,分别为0、2.5、5.0、10.0、20.0、40.0μg·mL~(-1),其中D+Q对照品组的药液按照WTI中D与Q的含量比例配制(D:Q=1:10),以相同的条件培养72hr,MTT法测定各孔吸光度,计算出各个浓度下的生长抑制率(IR),并求出各药的半数抑制率(IC_(50))。
3.2 WTI对正常腹膜与粘连腹膜成纤维细胞细胞周期的作用
取与3.1相同的NFB与AFB接种至培养皿中,均设空白、WTI、D对照品、Q对照品、D+Q对照品、地塞米松注射剂6个组,其中D+Q对照品组的药液按照10.0μg·mL~(-1)WTI中D与Q的含量比例配制(D:Q=1:10),其余药物处理组在相同培养条件下以10.0μg·mL~(-1)的药物浓度培养72hr,流式细胞术(FCM)检测各组FB的细胞周期。
3.3 WTI对正常腹膜与粘连腹膜成纤维细胞细胞超微结构的作用
取与3.1相同的NFB与AFB接种至培养皿中,设空白组与WTI组,其中WTI组的药物浓度为10μg·mL~(-1),在相同的条件下培养72hr,收集各组细胞,经固定、切片、染色等处理后,在透射电镜下观察其超微结构。
4 WTI对正常腹膜与粘连腹膜成纤维细胞目的基因mRNA表达与合成产物的作用研究
取与3.1相同的NFB与AFB,提取总RNA,利用内参照β-actin的引物与目的基因TGF-β_1、TGF-β_2、MMP-1、TIMP-1的引物进行RT-PCR。扩增产物经琼脂糖凝胶电泳分离后进行纯化,得到定量标准模板,按倍比稀释得到不同浓度的标准模板,加入相应的荧光探针与引物,在Real-time PCR仪上进行PCR扩增,将不同浓度的标准模板拷贝的对数和相应的C_T值(Threshold Cycle)作图,得到内参照与各目的基因的标准曲线。
取与3.1相同的NFB与AFB接种至培养皿中,均设空白、WTI、D对照品、Q对照品、D+Q对照品、地塞米松注射剂6个组,其中D+Q对照品组的药液按照10.0μg·mL~(-1)WTI中D与Q的含量比例配制(D:Q=1:10),其余药物处理组在相同培养条件下以10.0μg·mL~(-1)的药物浓度培养72hr,提取各组细胞的总RNA,逆转录为cDNA后,加入相应的荧光探针与引物,在Real-time PCR仪上进行PCR扩增,测定各组的C_T值,从相应目的基因标准曲线上求出各自的起始模板拷贝数,与内参照β-actin的拷贝数相比,求出各组FB不同目的基因(TGF-β_1、TGF-β_2、MMP-1、TIMP-1)的相对表达量。同时收集NFB与AFB各组培养72hr后的培养液,碱水解比色法测定HyP浓度,测定胶原合成水平。
结果
1 WTI对大鼠术后粘连模型的作用研究
1.1 WTI对大鼠粘连程度的影响
大鼠各实验组粘连程度均存在显著性差异(P＜0.05)。大鼠WTI高、低剂量组与地塞米松的平均秩次组均低于模型对照组,WTI高剂量组与地塞米松组的平均秩次也低于WTI低剂量组;
1.2 WTI对大鼠血液指标的影响
模型对照组WBC与其余各组有显著性差异(P＜0.05),各给药组WBC与正常对照组无显著性差异,各给药组之间WBC也无显著性差异(P＞0.05);WTI高剂量组RBC与地塞米松组有显著性差异(P＜0.05);各组HGB、LYM无显著性差异(P＞0.05);模型对照组PLT与WTI低剂量组无显著差异(p＞0.05),但与其余各组有显著性差异(P＜0.05),地塞米松组与正常对照组、WTI高剂量组均无显著差异(P＞0.05);模型对照组NEU与其余各组存在显著性差异(P＜0.05),其余各组间NEU无显著性差异(P＞0.05)。
1.3 WTI对大鼠盲肠壁组织HyP含量的影响
正常对照组HyP低于其余各组,并有显著性差异(P＜0.05);模型对照组HyP高于其余各组,也有显著性差异(P＜0.05);地塞米松组与WTI低剂量组无显著性差异(P＞0.05),与WTI高剂量组有显著性差异(P＜0.05)。
1.4大鼠病理切片观察
正常盲肠壁结构基本清晰,可见粘膜、粘膜下层、肌层、浆膜层四层结构。粘膜固有层内可见少量炎性细胞浸润,粘膜下层可见少量疏松结缔组织,肌层内环形及外纵层平滑肌结构清晰。浆膜层很薄,外部覆盖间皮细胞;模型对照组可见盲肠组织与结肠、小肠粘连,粘连带中有较多纤维组织增生并伴有充血及大量炎性细胞浸润,纤维组织甚至可见玻璃样变;WTI低剂量组可见盲肠组织中轻度粘连,纤维组织增生并伴有充血及炎性细胞浸润;WTI高剂量组可见部分盲肠组织中少量纤维组织增生并伴有散在炎性细胞浸润;地塞米松组与WTI高剂量组情况相似。
2 WTI主要成分的分析研究
测得WTI中D含量为4.99%,Q-A含量为14.98%,Q-B含量为11.42%,采用的色谱方法不受其它组分的干扰,精密度、回收率、稳定性良好,线性范围可满足测定的需要。
3 WTI对正常腹膜与粘连腹膜成纤维细胞增殖的作用研究
3.1 WTI对FB增殖活性的影响
AFB各药物处理组的IC_(50)均与NFB存在显著性差异(P＜0.05),WTI及其主要成分、地塞米松对AFB的IC_(50)值低于NFB。剂量-光密度曲线显示:AFB的WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的OD值下降幅度高于NFB各组。
3.2 WTI对FB细胞周期的影响
AFB空白组G_0/G_1期与NFB空白组无显著性差异(P＞0.05),G_2/M期与NFB空白组有显著差异(P＜0.05),其中S期细胞百分数高于NFB空白组,G_2/M期细胞百分数低于NFB空白组;AFB各药物处理组除地塞米松组外,G_0/G_1期、S期、G_2/M期均与NFB相应的药物处理组有显著性差异(P＜0.05),G_0/G_1期细胞百分数均高于NFB组,S期、G_2/M期细胞百分数均低于NFB组;AFB地塞米松组G_0/G_1期、G_2/M期与NFB地塞米松组有显著性差异(P＜0.05),S期与NFB地塞米松组无显著性差异(P＞0.05),其中G_0/G_1期细胞百分数高于NFB地塞米松组,G_2/M期细胞百分数低于NFB地塞米松组。
3.3 WTI对FB超微结构的影响
观察比较NFB与AFB未加药培养时的超微结构,未见有明显差异。当加入WTI培养后,AFB溶酶体数目增多,脂滴数目较多,内质网有扩张现象,线粒体有空泡变,嵴不明显。而NFB脂滴数目较少,溶酶体数目少,内质网正常,有明显的高尔基体存在。由此说明WTI可对NFB与AFB的超微结构产生影响,这应与FB增殖活性受到抑制有关。
4 WTI对正常腹膜与粘连腹膜成纤维细胞目的基因mRNA表达与合成产物的作用研究
4.1 TGF-β_1的相对表达量
AFB与NFB空白组的TGF-β_1 mRNA相对表达量有显著差异(P＜0.05);NFB的各个药物处理组均与空白组存在显著差异(P＜0.05);与空白组相比,WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的TGF-β_1mRNA相对表达量分别下降14.94%、8.11%、9.86%、10.33%、8.11%;AFB的各个药物处理组均与空白组存在显著差异(P＜0.05),与空白组相比,WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的TGF-β_1 mRNA相对表达量分别下降19.07%、26.69%、26.91%、16.14%、8.05%。
4.2 TGF-β_2的相对表达量
AFB与NFB空白组的TGF-β_2 mRNA相对表达量有显著差异(P＜0.05);NFB的各个药物处理组均与空白组存在显著差异(P＜0.05),与空白组相比,WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的TGF-β_1mRNA相对表达量分别下降17.02%、18.92%、33.45%、18.56%、8.98%;AFB的各个药物处理组均与空白组存在显著差异(P＜0.05),与空白组相比,WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的TGF-β_2 mRNA相对表达量分别下降40.47%、42.97%、49.24%、40.94%、20.41%。
4.3 MMP-1与TIMP-1的相对表达量
AFB与NFB空白组的MMP-1、TIMP-1 mRNA相对表达量均有显著差异(P＜0.05);NFB除地塞米松组的MMP-1与空白组无显著差异外(P＞0.05),其余各药物处理组均与空白组存在显著差异(P＜0.05);AFB的各个药物处理组均与空白组存在显著差异(P＜0.05)。
AFB与NFB空白组的MMP-1/TIMP-1比值存在显著性差异(P＜0.05),AFB的MMP-1/TIMP-1比值高于NFB;AFB各个药物处理组与AFB空白组均存在显著差异(P＜0.05),WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的MMP-1/TIMP-1比值分别增加14.74%、17.31%、21.79%、21.77%、11.54%;NFB的各个药物处理组与NFB空白组均无显著差异(P＞0.05)。
4.4胶原合成水平
AFB与NFB空白组的HyP存在显著性差异(P＜0.05),AFB的HyP高于NFB;AFB各个药物处理组与AFB空白组均存在显著差异(P＜0.05),WTI组、D对照品组、Q对照品组、D+Q对照品组和地塞米松组的HyP含量分别降低43.05%、39.68%、37.75%、36.75%、46.43%;NFB各个药物处理组与NFB空白组均无显著差异(P＞0.05)。
结论
1 WTI可减少腹膜受损处的胶原过度合成,降低该部位的纤维化程度,同时可减少炎症反应,降低过高的血液粘度,从而发挥粘连防治作用。
2 WTI的主要活性成分为D与Q。
3 WTI及其主要成分对FB增殖均有抑制作用,而且对AFB的作用比NFB强;同时还可调整FB的细胞周期,使其受阻于G_1期,延缓向S期、G2期过渡的速度,进而阻止FB进入M期,达到抑制其增殖的效果,对AFB调节作用也强于NFB。
4 WTI及其主要成分能有效降低AFB的TGF-β_1、TGF-β_2 mRNA表达水平,降低AFB的增殖速度,同时通过调节AFB的MMP-1与TIMP-1相对表达量比例,加快ECM降解速率,减少胶原合成量,从而减少粘连部位的纤维化程度。
Background
Post-surgical adhesion is a quite common complication,especially after general laparotomy.It manifests with fibrous bands joining normally separated organs together with the intestinal/abdominal wall.Intra-abdominal adhesion formation and re-formation after surgery is a significant cause of morbidity,such as pain,bowel obstruction,even infertility.According to previous studies,although recent cases suggests that a proportion of people suffering such adhesion may benefit from adhesiolysis,adhesion re-formation still remains as the most serious post-surgical problem,the probability ranging from 55%to 95%among the patients even those who have had undergone surgical adhesion removal.Up to now,a number of TCM (Traditional Chinese Medicine) treatment and herbal preparation have been evaluated for their value in post-surgical adhesion prevention.So it is necessary to exert the superiority and characteristic of TCM in treating post-surgical adhesion.
WTI(Weitong Injection),developed by Department of pharmaceutics,Nanfang Hospital,is a pure herbal injectable powder for anti-adhesion.It bases on the study of Changtong Oral Liqud and contains components from herb,which can improve microcirculation of blood,decrease inflammatory reaction and exudation.It is another important new drug for post-surgical adhesion.
Objective
To evaluate and explore the pharmacodynamic action and mechanism of WTI with the following experiments:To observe the effect of WTI on post-surgical adhesions by establishing rat experimental models with intestinal adhesions.To detect the main components of WTI by HPLC.To explore the effect of WTI and its components on proliferation and cell cycle of cultured fibroblasts from normal and adhesive peritoneal tissues.To detect synthesis product and mRNA expression of cytokines from NFB and AFB.
Methods
1.Pharmacodynamic action of WTI
Rats were received laparotomy and prepared as intestinal adhesion model.WTI was intravenously injected for 7 days.Dexamethasone were used as positive control. Blood routine examination was conducted.Intraperitoneal adhesions were graded by adhesion score on day 7.Tissues from normal and adhesive tissues were collected to prepare pathological sections.
2.Detection of components in WTI
Components of WTI were determined by HPLC.The mobile phase of D was phosphate buffer solution:methanol(95:5).The mobile phase of Q was 1% phosphoric acid:acetonitrile(60:40).Flow rate was 1mL·min~(-1).The UV detective wavelength was 220nm.Chromatographic column was Luna~(?) 5u C18(2) 100A. Column temperature was room temperature.
3.Effect of WTI on proliferation and cell cycle of NFB and AFB
The normal and adhesive peritoneal tissues were harvested from adhesion model of rots.The prepared tissues were cultured for 1～2 weeks until outgrowth of NFB and AFB.The cells of 3～5 passage were seeded on 96-well plates at a density of approximately 5×10~3 cells per well in 10%FBS.Then each kind of FB was divided into 5 groups:WTI,D standard,Q standard,D+Q standard and dexamethasone group. Each group had 6 different drug concentration ranging from 0～40μg·mL~(-1).The dosage of D+Q standard group was based on the ratio of D and Q in WTI(D:Q=1:10). Cultured for 72hr,the proliferation of NFB and AFB were detected by MTT assay.
The cells of 3～5 passage were seeded on culture dishes at a density of approximately 1×10~5 cells per dish in 10%FBS.Then each kind of FB was divided into 6 groups:blank,WTI,D standard,Q standard,D+Q standard and dexamethasone group.The dosage of D+Q standard group was based on the ratio of D and Q in 10μg·mL~(-1)WTI(D:Q=1:10).The drug concentration of other drug groups was 10μg·mL~(-1).Cultured for 72hr,the cell cycle of NFB and AFB were analyzed by FCM.
The cells of 3～5 passage were seeded on culture dishes at a density of approximately 1×10~5 cells per dish in 10%FBS.Then each kind of FB was divided into 2 groups:blank and WTI group.The drug concentration of WTI group is 10μg·mL~(-1).Cultured for 72hr,then cells were collected to observe the ultramicrostructure by transmission electron microscope.
4.Effect of WTI on cytokines mRNA expression and collagen synthesis of NFB and AFB
The cells of 3～5 passage were seeded on culture dishes at a density of approximately 1×10~5 cells per dish in 10%FBS.Then each kind of FB was divided into 5 groups:blank,WTI,D standard,Q standard,D+Q standard and dexamethasone group.The dosage of D+Q standard group was based on the ratio of D and Q in 10μg·mL~(-1)WTI(D:Q=1:10).The drug concentration of other drug groups was 10μg·mL~(-1).Cultured for 72hr,cells were collected to extract total RNA.Then the RT reaction was carried out on each RNA sample to produce cDNA.Real-time PCR was performed in a reaction plate containing RT products(cDNA),TaqMan fluorescent probe,relative primer and other PCR reagents.C_T of each sample was recorded. According to the standard curve ofβ-actin and each target gene,the copy number of original templates(β-actin,TGF-β_1,TGF-β_2,MMP-1,TIMP-1)were obtained.Finally, the absolute copy number of target gene was normalized to that ofβ-actin.On the other hand,collected the supernatant of cells and determine HyP by base hydrolysis-chromatometry.
Results
1.WTI could significantly reduce the level of adhesion,lower WBC and NEU counting,recover PLT counting to the preliminary level.WTI could also lower HyP concentration in traumatized tissues.Pathological sections showed the inflammation level of WTI groups were much lower than model group.Peritoneal and cecal wall of WTI groups were also fixed.Such effect of WTI were better than dexamethasone.
2.D and Q were the main components of WTI.The methods built up for HPLC were fit for the assaying of these components.The content of D was 4.99%.The content of Q-A and Q-B were 14.98%and 11.42%respectively.
3.IC_(50) of all AFB groups were lower than NFB groups.Dexamethosone groups had the highest IC_(50) among all AFB groups.Compared with blank groups,all drug groups had higher G_0/G_1 proportion and lower S,G_2/M proportion.Compared with all drug groups of NFB,all drug groups of AFB had higher G_0/G_1 proportion and lower S, G_2/M proportion.Transmission electron microscope picture showed WTI could change the ultramicrostructure of AFB.
4.Compared with blank group,the mRNA level of TGF-β_1,TGF-β_2,MMP-1, TIMP-1 of all drug groups were lower.Among the AFB groups,MMP-1/TIMP-1 mRNA ratio of drug groups were higher than blank group,and there was no significant difference between NFB drug groups and NFB blank group.Compared with blank groups of AFB,drug groups of AFB had lower HyP in the supernatant,and there was no significant difference between NFB drug groups and NFB blank group.
Conclusions
1.WTI could reduce excess synthesis of collagen in traumatized peritoneum and decrease fibrosis level.WTI could also reduce inflammatory reaction and improve the high blood viscosity caused by trauma.
2.D and Q were the main components of WTI.
3.Both WTI and its main components could regulate the cell cycle of FB and restrain the proliferation of FB.And AFB was more sensitive to medication than NFB.
4.Both WTI and its main components could lower mRNA expression level of TGF-β_1 and TGF-β_2 in AFB to restrain the proliferation of FB.They could also regulate MMP-1/TIMP-1 mRNA ratio in AFB to decrease synthesis of collagen,which lead to the inhibition of excess fibrosis.
引文
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[4]Saed GM,Diamond MP.Effect of glucose on the expression of type Ⅰ collagen and transforming growth factor-β_1 in cultured human peritoneal fibroblasts.Fertil Steril.2003;79(1):158-163
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