Abstract背景： 腹腔手术后造成肠粘连形成率约占90%, 而一旦肠粘连形成后则因肠粘连的原因需行二次手术的患者占5%-20%[2-4]。据估计在美国每年每10万人中有117例住院患者因术后肠粘连的原因二次住院，因二次住院的花费巨大，达数亿元[5, 6]。许多国家中患者因腹腔术后肠粘连的缘由其治疗费用已经超过原发疾病本身的治疗费用[5, 7, 8]。术后并发肠粘连后除了给患者带来沉重的经济负担外，同时也严重影响了其生活质量。肠粘连最常见的并发症是肠梗阻，反过来造成肠梗阻原因60%-70%是肠粘连所致[9, 10]。育龄期妇女中因盆腔手术后并发肠粘连造成的再孕障碍或不孕占15%-40%[11, 12]。肠粘连同时也是造成许多慢性腹痛的原因之一，此外因肠粘连的原因导致患者二次行腹腔镜手术难度极大，并且很大程度上增加了二次腹腔手术肠管等脏器的损伤机率[13, 14]。目前已被用于预防术后肠粘连形成的药物或材料较多，其在发挥抗粘连形成方面的效果均达不到理想目标，因此探索一种新的有效的预防肠粘连药物是非常有必要的。 目的： 通过芍药苷具有抗氧化应激（ROS 、SOD-2）、抑制炎症反应降低炎症因子（TGF-β1, IL-6以及COX-2)的表达从而减轻术后肠粘连的形成，降低术后创面纤维、胶原蛋白的沉积（MMP-9 , α-SMA and Collagen 1）及促进腹膜间皮细胞愈合（CK）,进一步为临床术后预防腹腔粘连形成提供参考。 方法： 选用雄性SD大鼠48只，随机分为组：正常对照组（Sham）、模型对照组（Control）、透明质酸钠组（HA）及三组芍药苷不同药物梯度组10 mg/kg (L-PE); 20 mg/kg (M-PE) 、40 mg/kg (H-PE)。除正常对照组外，其余各组实验动物均按盲肠摩擦+对应腹壁损伤模型构建肠粘连模型。每组大鼠术后均无限制食水喂养7天。正常对照组和模型组每天与药物组给予等量的生理盐水灌胃，连续7天；透明质酸钠组（HA）组术后腹腔给透明质酸钠1ml（40mg/kg）1次；三组芍药苷组每天灌胃，连续7天按照体重给药。各组于术后第8日在麻醉状态下行U型幵腹，留取腹腔渗液置于冻存管于液氮中保存，按肠粘连分级标准，肉眼宏观观察各组大鼠术后粘连分级情况；立即腹腔动脉穿刺抽血3-5ml于促凝管置冰盒，并进一步离心取血清后-80℃保存，留待酶联免疫吸附法（ELISA）检测各组大鼠血清标本中炎症因子的浓度；于大鼠肠粘连最显著处取大小约1.0cm×0.5cm×0.5cm的2块粘连组织，分别置于的中性甲醛固定液中及液氮罐中保存备用。中性甲醛固定24小时后常规石蜡包埋、切片，行各指标的免疫组化染色并评分、HE染色并光镜下观察成纤维细胞及炎症细胞浸润等情况。液氮保存的组织进一步行各指标的Western blotting检测；对于未形成粘连者分别取相同大小的受损盲肠壁与腹壁。 结果： 术后第8天，无死亡大鼠，每组动物体重在术前术后无明显改变。实验结果显示，宏观肠粘连评估情况：与正常对照组比较，模型对照组动物均发生粘连，且粘连程度较高，有大量的含血管片状粘连、粘连范围广泛而且紧密，粘连范围成程度和严重程度均评分均显著升高（P<0.05）。与Control组比较，HA组、L-PE组粘连程度均降低；M-PE与H-PE组粘连程度显著减轻；PE组肠粘连评分较对照组显著减低（P<0.05）。HE组织染色显示Sham组盲肠壁炎症细胞浸润少、成纤维细胞形成极少；Control组在粘连面可见大量的炎性细胞、成纤维细胞及毛细血管形成；HA组与L-PE组炎性细胞渗出及成纤维细胞形成相近，炎症评分均显著低于Control组（P<0.05）；M-PE与H-PE组中炎症细胞浸润、成纤维细胞显著降低，粘膜愈合完整，炎症评分均显著低于模型对照组（P<0.05）；ELISA检测血清各组炎症因子显示TGF-β1及IL-6在M-PE与H-PE组中均显著下降（P<0.05）；腹腔渗液中SOD、ROS的活性检测显示分别在H-PE和M-PE、H-PE组中较模型组均显著下降（P<0.05）；免疫组化结果显示，与Contro组相比，SOD-2在PE组中显著升高；α-SMA、Collagen I在PE组中均显著下降，表明纤维形成水平下降，且与随着药物浓度的增加其染色越轻；反映胶原形成的天狼猩红染色在PE组中显著低于Control组（P<0.05）；上皮愈合染色中CK显示，在M-PE及H-PE组中创面腹膜间皮细胞上皮愈合完整，在Control组中创面间皮细胞结构紊乱；Western blotting 检测SOD-2, COX-2, MMP-9,?α-SMA的表达水平，结果显示与免疫组化结果相一致，COX-2在H-PE组中显著降低。 结论： 1. 芍药苷通过降低氧化应激和炎症反应机制减少术后创面纤维形成； 2. 芍药苷可能通过促进局部粘连组织的SOD-2表达增加组织抗氧化减轻了术后腹腔粘连； 3. 芍药苷可能通过降低组织炎症渗出、TGF-β1、IL-6以及COX-2的表达抑制炎症反应减轻了术后腹腔粘连，且随着药物浓度的增加效果更加明显即呈现出剂量依赖关系。
Background: The rate of peritoneal adhesions caused by abdominal surgery is more than 90%. And the range of the patients who needed secondary surgery is 5%-20% when once the intestinal adhesions formed. It is estimated that there are 117 hospitalized patients per 100,000 people who costs more than a hundred million due to secondary hospitalization in the United States each year for reasons of postoperative intestinal adhesions. In many countries, the cost of treatment due to postoperative abdominal adhesions has exceeded the cost of treatment for primary disease itself. Postoperative intestinal adhesion in addition to bring the heavy economic burden to patient , but also seriously affects the quality of life. The most common complication of intestinal adhesion is intestinal obstruction, in turn, about 60% -70% of intestinal obstruction caused by intestinal adhesion. It is difficult to re-pregnancy or infertility when intestinal adhesion formation after pelvic surgery for the pregnancy women. Intestinal adhesions are also one of the causes of many chronic abdominal pain, in addition, it is difficult to perform the secondary laparoscopic surgery for the patients who have formed the intestinal adhesions, and to a large extent it pancreas the rate of intestine and other organs injury when implement the secondary abdominal surgery. Drugs or materials in currently not recognized, but used to prevent the formation of postoperative intestinal adhesion are abundant. All these targets play in the anti-adhesion effect are not ideal, so it is necessary to explore a new effective drug to prevention of intestinal adhesions. Objective: Paeoniflorin prevents postoperative peritoneal adhesion formation via anti-oxidative stress (ROS, SOD-2)and anti-inflammatory(TGF-β1, IL-6 and COX-2), reducing postoperative wound fibers, collagen deposition ( MMP-9, α-SMA and Collagen 1) and promoting the healing of peritoneal mesothelial cells (CK), and further provide reference for the prevention of intraperitoneal adhesions after clinical operation. Methods: Forty-eight Sprague-Dawley male rats (weight, 180200 g) were obtained from the Animal Resource Center of Xi’an Jiaotong University. The rats were kept in cages under standard conditions and were allowed free access to food and water. The guidelines of the Animal Care and Use Committee of the Xi’an Jiaotong University Health Science Center served as the reference standard. Our experimental procedures and processes were approved by the Ethics Committee. The rats were randomly divided into groups: the sham group, the control group, the ?hyaluronate(HA) group and three paeoniflorin(PE) groups. The PE divided into three subgroups, 10 mg/kg (L-PE); 20 mg/kg (M-PE) 、40 mg/kg (H-PE) respectively. The animals were anesthetized with a 50 mg/kg peritoneal injection of pentobarbital for all procedures. Prior to incision, the abdomen was shaved, and the skin was sterilized with antiseptics. An approximately 2 cm incision of the abdominal wall was made for each animal. The anterior cecal surface was gently abraded 30 times to the same extent with a wet swab until partial petechial hemorrhages were generated. The abdominal wall that faced the treated cecum was damaged using a medical electric scalpel over an area of approximately 2 cm2. The cecum was then placed back in its original location. In the sham operation group, cecal abrasion was not performed, and the abdominal wall was not damaged. In the hyaluronan (HA) group, 1 mL HA gel was applied on two sides of the trauma and its surrounding area. The control group received 1 mL normal saline, and the three PE groups received low-dose PE (dissolved in normal saline; purity >98%, Sigma-Aldrich Co. LLC., St. Louis, MO, USA; ) at 10 mg/kg (L-PE); moderate-dose PE at 20 mg/kg (M-PE) or high-dose PE at 40 mg/kg (H-PE) via gavage for 7 days. The abdominal incision was closed in two layers with a continuous 3/0 silk suture. After 7 days, all the animals were survived. A U-shaped incision was made to estimate the adhesions and take specimens. Immediately, aortaventralis blood about 3-5ml into the evacuated?and promoting coagulating?tubes and then placed in the ice box. After the tubes centrifuged for 15mins the serum preserved at -80 ℃. The enzyme-linked immunosorbent assay (ELISA) were used to detect the concentration of inflammatory cytokines in each group of serum samples. In the obvious intestinal adhesions, about 1.0cm × 0.5cm × 0.5cm of the two adherent tissue were token and fixed in the neutral formalin fixative and liquid nitrogen for the following test. The ?tissues after 24 hours of fixed with neutral formalin, then paraffin-embedded, sliced and immunohistochemical staining of each index were performed. The expression of fibroblasts and inflammatory cells were observed by HE staining and light microscopy. The ?tissues that preserved in liquid nitrogen were tasted further the indicators by western blotting; as for the rats who did not form of adhesions, the specimen were token from the damaged cecal wall and opposite abdominal wall. Result: After 7 days of feeding, all rats survived, and no significant differences were found in the body weights of the rats in the six groups. The degree of abdominal adhesion in each group was lower than that of the control group. In the sham group, there was only a small amount of adhesion near the peritoneal incision, and intestine-to-abdominal-wall adhesions were relatively uncommon. In contrast, the control group exhibited numerous omentum-to-bowel, bowel-to-parietal peritoneum, parietal peritoneum-to-omentum, and bowel-to-bowel adhesions. The three groups of rats that had been treated with PE had fewer peritoneal adhesions than the control group. The PE groups had the lowest adhesion scores in all of the groups. The H-PE group had less-severe adhesions. There was a clear difference between the PE groups and the control group. For the HA group, the intestinal adhesions were also minimal. HE staining showed that the infiltration of inflammatory cells in the cecal wall of sham group was few, and fibroblast formation was minimal. In the control group, showed a ?abundant of inflammatory cells, fibroblasts and capillaries on the adhesive tissues. In the HA group and L-PE group, it was parallel?to the inflammatory cell exudation and fibroblast formation, the inflammation score was significantly lower than the control group (P <0.05). In the M-PE and H-PE groups, the fibroblasts were significantly decreased, the mucosal healing was complete and the inflammation score was significantly lower than that of the model control group (P <0.05). The levels of TGF-β1 and IL-6 in M-PE and H-PE groups were significantly decreased by ELISA (P <0.05). The activities of SOD-2 and ROS in enterocoelic fluid specimens showed a significant decrease in H-PE, M-PE and H-PE groups separately (P <0.05). The immunohistochemical staining results showed, compared with the control group, SOD-2 was significantly increased in three PE groups; α-SMA and collagen I decreased in the three PE groups, significantly in the M-PE and H-PE groups indicating that the fiber formation level decreased, and with the increase in drug concentration, the lighter the staining. The maker of collagen picrosirius red staining in the PE groups were significantly lower than that in the control group (P <0.05). The mesothelial cell continuity and the repair of the cell layer of the damaged rat peritoneum were further evaluated. The expression of cytokeratin was detected via immunohistochemical staining. In the sham group, the results showed a complete and continuous cell layer on the surface of the two-layer peritoneum (parietal and visceral). In contrast, relatively few mesothelial cells were present on the surface of the damaged two-layer peritoneum in the control group. In the HA and L-PE group, mesothelial cells were equivalent and inconspicuous. Finally, an abundant and a well-repaired mesothelial layer was apparent in the M-PE and H-PE groups. In the H-PE group, mesothelial cell repair was more complete and continuous than in the M-PE group. The expression of SOD-2, COX-2, MMP-9 and α-SMA were detected by western blotting. The results showed that COX-2 was significantly decreased in M-PE and H-PE groups, the others were equivalent to the immunohistochemical staining. Conclusion: 1. PE reduces the postoperative fibrous formation by inhibiting oxidative stress and inflammatory reaction mechanism. 2. PE may increase tissue antioxidant by promoting the expression of SOD-2 in local adhesion tissue to reduce postoperative abdominal adhesions. 3. PE may reduce the adhesion by inhibition of inflammatory response with decreasing the inflammatory cells exudated into the tissue, TGF-β1, IL-6 and COX-2 expression. With the increase of drug concentration, the effect is more obvious, and the PE markedly suppressed postoperative peritoneal adhesions in a dose-dependent manner in a rat model.