Original Papers
969
Authors
Chengxi Wang 1, 2, Xiao Li 2, Xiao Meng 2, Jing Zhou 3, Fei Qin 2, Lianbing Hou 2
Affiliations
1 2 3
Key words " tanshinone IIA l " peritoneal adhesions l " tPA l " PAI‑1 l " inflammatory response l " peritoneal fibrinolysis l " COX‑2 l
received revised accepted
May 8, 2014 June 9, 2014 June 16, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382877 Published online August 4, 2014 Planta Med 2014; 80: 969–973 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Lianbing Hou Nanfang Hospital, Southern Medical University Department of Pharmaceutical Science No. 1838, North of Guangzhou Avenue Guangzhou 510515 China Phone: + 86 0 20 61 64 21 75 [email protected]
Department of Pharmacology, Guangdong Pharmaceutical University, Guangzhou, China Department of Pharmaceutical Science, Nanfang Hospital, Southern Medical University, Guangzhou, China Department of Pharmacology, Inner Mongolia Medical University, Hohhot, China
Abstract !
Postoperative adhesions develop after nearly every abdominal surgery. The formation of adhesions is associated with the inflammatory response, fibrinolytic system, and extracellular matrix deposition in response to injury. Tanshinone IIA is one of the major extracts obtained from Salvia miltiorrhiza, which has anti-inflammatory effects on many diseases. Postoperative adhesions were induced by injuring the parietal peritoneum and cecum in Wistar rats, followed by the administration of various dosages of tanshinone IIA. The adhesion scores for each group were collected seven days after the initial laparotomy. The activity of the tissue-type plasminogen activator in the peritoneal lavage fluid was measured. The messenger ribonucleic acid expression levels of the tissue-type plasminogen activator, plasminogen activator inhibitor-1, and cyclooxygenase-2 in the ischaemic tissues were measured by quantitative real-time polymerase chain reaction. The intraperitoneal administration of tanshinone IIA is effective for the prevention of the formation of postoperative adhesions in rats. Tanshinone IIA increased fibrinolytic activity in the peritoneal
Introduction !
Adhesions are the most frequent complication of abdominal surgery, and a considerable proportion of cases result in major short- and long-term negative consequences, including small-bowel obstruction, infertility, and chronic pelvic pain [1]. After laparotomy, adhesions are found in nearly 93% of patients during subsequent surgeries [2]. Many patients require repeated surgical treatments, which place a substantial burden on the health care system and individuals. Studies have shown that depositions of a fibrinrich matrix on peritoneal surfaces can physically
lavage fluid and tissue-type plasminogen activator messenger ribonucleic acid expression in ischaemic peritoneal tissues but decreased the plasminogen activator inhibitor and cyclooxygenase-2 messenger ribonucleic acid expression significantly. These results revealed that tanshinone IIA was a potent postoperative adhesion preventer by enhancing fibrinolytic activity and decreasing cyclooxygenase-2 activity.
Abbreviations !
COX-2: DEX: HD: LD: NSAID: PAI: PPA: qRT‑PCR: TanIIA: tPA: uPA:
cyclooxygenase-2 dexamethasone high dose low dose nonsteroidal anti-inflammatory drug plasminogen activator inhibitor postoperative adhesions quantitative real-time polymerase chain reaction tanshinone IIA tissue-type plasminogen activator urokinase-type plasminogen activator
interact, form bands containing fibroblasts, and result in permanent adhesions after surgery [3]. The peritoneal fibrinolytic system is responsible for eliminating the fibrin-rich matrix via two plasminogen activators: tPA and uPA [4]. Because uPA is limited in its capacity to activate plasminogen, tPA is the primary plasminogen activator in the peritoneum [5, 6]. One cause of adhesion formation is an inflammatory response. The extent of adhesion formation is determined by the degree of peritoneal inflammation [7]. The inflammatory response, which is normally induced by infection or tissue injury, is crucial in promoting wound healing [8]. Several
Wang C et al. Prevention of Experimental …
Planta Med 2014; 80: 969–973
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Prevention of Experimental Postoperative Peritoneal Adhesions through the Intraperitoneal Administration of Tanshinone IIA
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Original Papers
anti-inflammatory agents have been shown to prevent adhesion formation [9]. " Fig. 1), a derivative of phenanthrenequinone, is one of TanIIA (l the key components of Danshen, which is derived from the dried root of Salvia miltiorrhiza Bunge (Lamiaceae) and has been widely used for centuries in many Asian countries for the prevention and management of cardiovascular diseases and various microcirculatory disturbance-related diseases [10]. As a main component of Danshen, TanIIA has exhibited the ability to attenuate inflammatory responses in rats with myocardial infarction [11–13]. Studies have demonstrated that TanIIA facilitates anti-inflammatory activities primarily by suppressing the transcription of proinflammatory cytokines [14]. Furthermore, previous in vitro studies have indicated that TanIIA effectively prevents the proliferation of fibroblasts derived from adhesion tissues in the rat [15]. In summary, this study investigates the effects of TanIIA on the formation of postoperative peritoneal adhesions to assess whether TanIIA can improve postoperative peritoneal wound healing.
Fig. 2 Effects of TanIIA tissue-type plasminogen activator activity in intraperitoneal lavage fluid. Both DEX (n = 15) and high-dose-treated animals (n = 15) showed a significant increase in tPA activity in peritoneal fluid compared with the LD treated (n = 15) and vehicle (n = 15). Results are expressed as mean ± SEM. *P < 0.05 compared with the vehicle, #p < 0.05 compared with the LD of TanIIA (2.5 mg/kg).
Results !
The scores for the intra-abdominal adhesions on postoperative " Table 1. The intraperitoneal adday 7 of rats are summarised in l ministration of the HD of TanIIA or DEX significantly decreased the adhesion formation. Conversely, every vehicle-treated animal developed adhesions to some degree. Compared with the vehicletreated animals, the HD group exhibited a 68% reduction in adhesion formation, and the LD group exhibited a 38% reduction. The LD group showed significantly lower adhesion scores than the HD and DEX groups, and the adhesion scores of the vehicle group were significantly lower than those found for all of the drug-treated (HD, LD, and DEX) groups. After assessing the adhesion in the abdomen, we collected the peritoneal fluid of the rats to detect the tPA activity in the peritoneum. The administration of TanIIA increased tPA activity in the peritoneal lavage fluid seven days " Fig. 2). The tPA activity levels of both the HD after surgery (l (19.65 ± 2.63) and DEX (20.65 ± 2.63) groups were significantly (p < 0.05) higher than those of the vehicle (6.75 ± 1.54) and LD (11.67 ± 2.39) groups. Meanwhile, the activity level of the LD group was higher than that of the vehicle group (p < 0.05). When the animals were sacrificed, the ischaemic button tissues in the abdomen of each rat were collected. The total RNA of the ischaemic button tissues was isolated and analysed by qRT‑PCR, and the results showed a significant (p < 0.05) increase in the tPA mRNA expression levels in the peritoneal tissues of the TanIIAtreated animals. Compared with those in the vehicle group, the animals treated with an HD of TanIIA presented a tPA expression level approximately six times greater, and those treated with an
" Fig. 3 A). In contrast, LD approximately three times greater (l the PAI-1 mRNA expression levels in the drug-treated groups were significantly (p < 0.05) decreased compared with those found in the vehicle group, and there were no significant differ" Fig. 3 B). The analyences between these drug-treated groups (l sis of COX-2 expression revealed that the HD of TanIIA reduced the COX-2 expression level to 60 % of the levels recorded in the vehicle-treated animals (p < 0.05). Similarly, the LD of TanIIA reduced the level of COX-2 mRNA to 82 % of that found in the vehicle-treated animals (p < 0.05). Altogether, TanIIA strongly " Fig. 3 B). downregulates COX-2 expression (l
Discussion !
TPA plays an important role in the peritoneal fibrinolytic system. Studies in humans have shown that decreased tPA activity or the overexpression of PAI-1 leads to more severe adhesions [16]. Sev-
Table 1 Intra-abdominal adhesion scores of groups.
a
Adhesion score
DEX
High dose of TanIIA
Low dose of TanIIA
Vehicle
0 1 2 3 4 Mean ± SD
6* 4 4 1 0 1.00 ± 1.00a, b
5 6 3 1 0 1.00 ± 0.93a, b
2 3 5 4 1 1.93 ± 1.16a
1 1 2 6 6 3.13 ± 0.92b
1 2 3 4 5
P < 0.05 compared with the vehicle group; bp < 0.05 compared with the LD of TanIIA group; * number of animals
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Fig. 1 Chemical structure of TanIIA.
Fig. 3 TpA (A), PAI-1, and COX-2 (B) mRNA expression in peritoneal tissues. Relative expression levels of tPA, PAI-1, and COX-2 mRNAs are normalised against β-actin. Results are expressed as a percent of vehicles and shown as mean ± SEM (n = 5). * P < 0.05 compared with the vehicle, # p < 0.05 compared with the LD of TanIIA (2.5 mg/kg).
eral chemicals, such as NK-1RA, statins, octreotide, and methylene blue, have been proven to be effective for the prevention of PPA formation by promoting tPA activity and upregulating peritoneal fibrinolysis [17–19]. In our experiments, it was found that on postoperative day 7, the tPA activity level in the peritoneal lavage fluid was increased significantly in drug-treated groups. This suggested that TanIIA could increase the activity of the peritoneal fibrinolytic system and effectively decrease adhesion formation in the abdomen. Furthermore, we also found that the mRNA ex-
pression level of tPA increased in the parietal peritoneal tissue of the TanIIA-treated animals. In contrast, TanIIA significantly de" Fig. 4 B). The PAI-1 procreased the PAI mRNA expression level (l tein, which appears to be an acute-phase reactant protein [20], is the most important inhibitor of tPA and uPA [21, 22]. Segura et al. demonstrated that the RNA interference-mediated downregulation of the PAI-1 gene in rats can prevent abdominal adhesion formation [23]. The qPCR results discussed in this study correlate with the observed tPA activation in the peritoneal lavage fluid, which indicates that the upregulation of tPA mRNA expression and the downregulation of PAI mRNA can evoke tPA activation in the peritoneal cavity. Overall, these changes result in the upregulation of the fibrinolytic system. In addition to the PAI mRNA expression level, we found that the COX-2 mRNA expression level was also significantly downregulated both in TanIIA-treated and DEX-treated animals compared with the vehicle-treated animals. It has been confirmed that anti-inflammatory drugs, including NSAIDs such as aspirin, can decrease adhesion formation in animals [24]. Anti-inflammatory agents have been used to reduce the initial inflammatory response to tissue injury [25]. As one of the most important proinflammatory mediators, COX-2 has been found to induce proinflammatory, proangiogenic, and profibroblastic effects, all of which may promote adhesion formation. Inhibition of the COX-2 enzyme, which is expressed by adhesion fibroblasts [26], has been shown to reduce adhesion formation in rodents [27–29]. The marked reduction of COX-2 mRNA expression by TanIIA suggests a second mechanism for the prevention of adhesion formation after abdominal surgery. It has been found that TanIIA, by decreasing COX-2 expression induced by lipopolysaccharides, inhibits prostaglandin E2 synthesis in osteoblasts, which, in turn, suppresses inflammatory bone loss [30]. Two recent studies have shown that TanIIA has a protection effect on the cells which are located in ischaemia areas, such as in cases of myocardial ischaemia and cerebral ischaemia in rats [31, 32]. Another study has demonstrated that the cardioprotective effects of TanIIA might be attributed to its capacity for inhibiting inflammatory responses. In an in vitro study, TanIIA was found to attenuate the myocardial infarction pathological changes, improve heart function, and reduce the expression of MCP-1, TGF-β1, and macrophages in filtration [12]. It may be concluded that there is a possibility that TanIIA be applied to PPA diseases because it significantly inhibits PPA, mainly through the decreasing inflammatory response process and activating the fibrinolysis system. Further research needs to be carried out on plasma-derived mediators such as C3, plasmin, and factor XII as well as cell-derived mediators such as histamine, IFN, and IL so as to clarify the mechanism of TanIIA in reducing inflammatory response.
Fig. 4 A Creation of the ischaemic buttons to the peritoneal sidewall as previously described. B Adhesions to the buttons at postoperative day 7. (Color figure available online only.)
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Original Papers
Original Papers
Materials and Methods
Table 2 Grading of adhesions in rats according to the criteria of Nair [41].
!
Animal model
Grade
Description
Classification
Male Wistar rats weighing 175 to 200 g, which were obtained from the Laboratory Animal Centre of Southern Medical University (Guangzhou, China), were used for all of the experiments. The rats were housed at a constant room temperature with 12-h light/12-h dark cycles and were provided with standard rodent chow and water ad libitum. All of the procedures and animal care protocols were approved by the Committee on the Ethics of Animal Experiments of Southern Medical University (permit number: 44 020 101 922, date: 04/22/2012). The abdominal hair of the rats was shaved and the animals were anaesthetised through the administration of 5% pentobarbital sodium by injection into the right side of the abdomen. After the lower abdomen was shaved and cleansed with 75% alcohol, a 3to 4-cm midline laparotomy was performed. Four ischaemic buttons, spaced 1 cm apart, were created on both sides of the parietal peritoneum by grasping 5 mm of the peritoneum with a haemostat and ligating the base of the segment with a 4–0 silk suture. The cecum was then isolated and scratched with a scalpel [33]. All of the surgical procedures were performed under sterile conditions. As the DEX has long been proven as an effective reference drug in preventing PPA [34–39], it was administrated to the control group as a reference drug. Sixty rats were divided into four equal groups (n = 15 per group). Each of the groups was administrated DEX (10 mg/kg, 99 % purity, Shanghai Yansheng Industrial Co., Ltd.), vehicle (100 % DMSO), and different amounts of TanIIA (98% purity, Xiʼan Honson Biotechnology Co., Ltd.), which were categorised as HD (10 mg/kg) and LD (2.5 mg/kg). In all groups, the intraperitoneal administration was performed at the time of laparotomy [40].
0
Complete absence of adhesions
Insubstantial adhesions
1
Single band of adhesions between viscera or from one viscus to abdominal wall Two bands, either between viscera or from viscera to the abdominal wall More than two bands between viscera or viscera to the abdomimal wall, or whole of intestines forming a mass without being adherent to the abdominal wall Viscera directly adherent to the abdominal wall, irrespective of number and extent of adhesive bands
Grading of adhesions in rats On postoperative day 7, an abdominal incision was made using the previously described anaesthetic method. The adhesions " Fig. 4) were graded as described by Nair et al. [41], and the ad(l hesion scores were determined by an investigator blind to the di" Table 2). vision of the groups (l
The tissue-type plasminogen activator activity in the peritoneal lavage fluid The peritoneal fluid was collected by rinsing the peritoneal cavity with 2 mL of phosphate buffer containing heparin (1 IU/mL, pH = 7.4) at 37 °C. Approximately 1 mL of the fluid was recovered and added to an equal volume of 0.2 M sodium acetate, pH 3.9. The cellular debris was then removed by microcentrifugation (2000 rpm × 1 min, 4 °C) [42]. The fibrinolytic activity in each sample due to tPA was assayed in duplicate by adding 50 µL of the diluted sample to different wells of a 96-well microtitre plate containing 50 µL of tPA stimulator (0.6 mg/mL cyanogen bromide-digested fibrinogen, American Diagnostica, Inc.). Then, 150 µL of assay buffer [16.7 µg/mL human plasminogen (Athens Research and Technologies), 667 µM S-2251 substrate (Chromogenix), and 20 mM Tris, pH 8.3] was added to each well and gently mixed. Cleavage of the S-2251 substrate by tPA-activated plasmin produced a yellow colour that absorbs wavelengths of 405 and 490 nm. The change in absorbance was measured at 37 °C over a 6-h period using a Spectra Max 250 spectrophotometer. The activity of tPA in each sample was determined by extrapolation from a tPA (human; Prospec) standard curve [42].
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2 3
4
Substantial adhesions
Detection of tissue-type plasminogen activator, plasminogen activator inhibitor, and cyclooxygenase-2 messenger ribonucleic acid expression levels by quantitative real-time polymerase chain reaction analysis The ischaemic button tissue of the parietal peritoneum, including a 1-mm rim of the surrounding tissue, was removed, frozen in liquid nitrogen, and stored at − 80 °C until qRT‑PCR analysis. The rats were then euthanised by a combination of pneumothorax and cardiac puncture. The ischaemic button tissues were homogenised by pulverisation in liquid nitrogen, and 1 mL of 0.1% Triton X 100 was added for every 40 mg of tissue. The total RNA from the peritoneal ischaemic button tissue was isolated according to the manufacturerʼs instructions (RNAiso Plus, Takara Biotechnology). The RNA concentrations were determined spectrophotometrically by measuring the absorbances at 260 and 280 nm, and the RNA quality was assessed through gel electrophoresis. The complementary DNA (cDNA) was synthesised from the total RNA using the All-inone™ first-strand cDNA synthesis kit (Genecopoeia), according to the manufacturerʼs suggested protocol, and qRT‑PCR was conducted using the All-in-one™ qPCR mix system (Genecopoeia) with SYBR green, according to the standard protocol. The following primer sets were used to amplify the following probes: " tPA, (F) 5′-CGCTGTACCTCACAGCATCTGTTTA‑3′ and (R) 5′-CATCCGCTTATCGATCATGCAC‑3′; " PAI-1, (F) 5′-ACCATCTCCGTGCCCATGA‑3′ and (R) 5′-GGGCAGTTCCAGGATGTCGTA‑3′; " COX-2, (F) 5′-AACACGGACTTGCTCACTTTGTTG‑3′ and (R) 5′-AATGGAGGCCTTTGCCACTG‑3′; " β-actin, (F) 5′-GGAGATTACTGCCCTGGCTCCTA‑3′ and (R) 5′-GACTCATCGTACTCCTGCTTGCTG‑3′. qRT‑PCR was performed using an Agilent Stratagene 3005P system. All PCRs were performed under the conditions recommended by the manufacturer: 95 °C for 10 min for the initial denaturation, 40 cycles of 95 °C for denaturation, 60 °C for 20 s for annealing, and 72 °C for 20 s for extension.
Statistical analysis The SPSS for Windows 10.0 software was used for the statistical analyses. The Kruskal-Wallis test was used to identify the differences in the adhesion grades between the groups. In addition, the Mann-Whitney U test was used to determine the differences be-
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tween each pair of groups. One-way ANOVA was performed to assess all of the other differences between the groups. If the differences were significant (p < 0.05), the Student-Newman-Keuls test was conducted to detect the differences between specific means. The results were evaluated at a significance level of p < 0.05.
Conflict of Interest !
The authors declare no conflict of interest.
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969
Authors
Chengxi Wang 1, 2, Xiao Li 2, Xiao Meng 2, Jing Zhou 3, Fei Qin 2, Lianbing Hou 2
Affiliations
1 2 3
Key words " tanshinone IIA l " peritoneal adhesions l " tPA l " PAI‑1 l " inflammatory response l " peritoneal fibrinolysis l " COX‑2 l
received revised accepted
May 8, 2014 June 9, 2014 June 16, 2014
Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1382877 Published online August 4, 2014 Planta Med 2014; 80: 969–973 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Prof. Lianbing Hou Nanfang Hospital, Southern Medical University Department of Pharmaceutical Science No. 1838, North of Guangzhou Avenue Guangzhou 510515 China Phone: + 86 0 20 61 64 21 75 [email protected]
Department of Pharmacology, Guangdong Pharmaceutical University, Guangzhou, China Department of Pharmaceutical Science, Nanfang Hospital, Southern Medical University, Guangzhou, China Department of Pharmacology, Inner Mongolia Medical University, Hohhot, China
Abstract !
Postoperative adhesions develop after nearly every abdominal surgery. The formation of adhesions is associated with the inflammatory response, fibrinolytic system, and extracellular matrix deposition in response to injury. Tanshinone IIA is one of the major extracts obtained from Salvia miltiorrhiza, which has anti-inflammatory effects on many diseases. Postoperative adhesions were induced by injuring the parietal peritoneum and cecum in Wistar rats, followed by the administration of various dosages of tanshinone IIA. The adhesion scores for each group were collected seven days after the initial laparotomy. The activity of the tissue-type plasminogen activator in the peritoneal lavage fluid was measured. The messenger ribonucleic acid expression levels of the tissue-type plasminogen activator, plasminogen activator inhibitor-1, and cyclooxygenase-2 in the ischaemic tissues were measured by quantitative real-time polymerase chain reaction. The intraperitoneal administration of tanshinone IIA is effective for the prevention of the formation of postoperative adhesions in rats. Tanshinone IIA increased fibrinolytic activity in the peritoneal
Introduction !
Adhesions are the most frequent complication of abdominal surgery, and a considerable proportion of cases result in major short- and long-term negative consequences, including small-bowel obstruction, infertility, and chronic pelvic pain [1]. After laparotomy, adhesions are found in nearly 93% of patients during subsequent surgeries [2]. Many patients require repeated surgical treatments, which place a substantial burden on the health care system and individuals. Studies have shown that depositions of a fibrinrich matrix on peritoneal surfaces can physically
lavage fluid and tissue-type plasminogen activator messenger ribonucleic acid expression in ischaemic peritoneal tissues but decreased the plasminogen activator inhibitor and cyclooxygenase-2 messenger ribonucleic acid expression significantly. These results revealed that tanshinone IIA was a potent postoperative adhesion preventer by enhancing fibrinolytic activity and decreasing cyclooxygenase-2 activity.
Abbreviations !
COX-2: DEX: HD: LD: NSAID: PAI: PPA: qRT‑PCR: TanIIA: tPA: uPA:
cyclooxygenase-2 dexamethasone high dose low dose nonsteroidal anti-inflammatory drug plasminogen activator inhibitor postoperative adhesions quantitative real-time polymerase chain reaction tanshinone IIA tissue-type plasminogen activator urokinase-type plasminogen activator
interact, form bands containing fibroblasts, and result in permanent adhesions after surgery [3]. The peritoneal fibrinolytic system is responsible for eliminating the fibrin-rich matrix via two plasminogen activators: tPA and uPA [4]. Because uPA is limited in its capacity to activate plasminogen, tPA is the primary plasminogen activator in the peritoneum [5, 6]. One cause of adhesion formation is an inflammatory response. The extent of adhesion formation is determined by the degree of peritoneal inflammation [7]. The inflammatory response, which is normally induced by infection or tissue injury, is crucial in promoting wound healing [8]. Several
Wang C et al. Prevention of Experimental …
Planta Med 2014; 80: 969–973
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Prevention of Experimental Postoperative Peritoneal Adhesions through the Intraperitoneal Administration of Tanshinone IIA
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Original Papers
anti-inflammatory agents have been shown to prevent adhesion formation [9]. " Fig. 1), a derivative of phenanthrenequinone, is one of TanIIA (l the key components of Danshen, which is derived from the dried root of Salvia miltiorrhiza Bunge (Lamiaceae) and has been widely used for centuries in many Asian countries for the prevention and management of cardiovascular diseases and various microcirculatory disturbance-related diseases [10]. As a main component of Danshen, TanIIA has exhibited the ability to attenuate inflammatory responses in rats with myocardial infarction [11–13]. Studies have demonstrated that TanIIA facilitates anti-inflammatory activities primarily by suppressing the transcription of proinflammatory cytokines [14]. Furthermore, previous in vitro studies have indicated that TanIIA effectively prevents the proliferation of fibroblasts derived from adhesion tissues in the rat [15]. In summary, this study investigates the effects of TanIIA on the formation of postoperative peritoneal adhesions to assess whether TanIIA can improve postoperative peritoneal wound healing.
Fig. 2 Effects of TanIIA tissue-type plasminogen activator activity in intraperitoneal lavage fluid. Both DEX (n = 15) and high-dose-treated animals (n = 15) showed a significant increase in tPA activity in peritoneal fluid compared with the LD treated (n = 15) and vehicle (n = 15). Results are expressed as mean ± SEM. *P < 0.05 compared with the vehicle, #p < 0.05 compared with the LD of TanIIA (2.5 mg/kg).
Results !
The scores for the intra-abdominal adhesions on postoperative " Table 1. The intraperitoneal adday 7 of rats are summarised in l ministration of the HD of TanIIA or DEX significantly decreased the adhesion formation. Conversely, every vehicle-treated animal developed adhesions to some degree. Compared with the vehicletreated animals, the HD group exhibited a 68% reduction in adhesion formation, and the LD group exhibited a 38% reduction. The LD group showed significantly lower adhesion scores than the HD and DEX groups, and the adhesion scores of the vehicle group were significantly lower than those found for all of the drug-treated (HD, LD, and DEX) groups. After assessing the adhesion in the abdomen, we collected the peritoneal fluid of the rats to detect the tPA activity in the peritoneum. The administration of TanIIA increased tPA activity in the peritoneal lavage fluid seven days " Fig. 2). The tPA activity levels of both the HD after surgery (l (19.65 ± 2.63) and DEX (20.65 ± 2.63) groups were significantly (p < 0.05) higher than those of the vehicle (6.75 ± 1.54) and LD (11.67 ± 2.39) groups. Meanwhile, the activity level of the LD group was higher than that of the vehicle group (p < 0.05). When the animals were sacrificed, the ischaemic button tissues in the abdomen of each rat were collected. The total RNA of the ischaemic button tissues was isolated and analysed by qRT‑PCR, and the results showed a significant (p < 0.05) increase in the tPA mRNA expression levels in the peritoneal tissues of the TanIIAtreated animals. Compared with those in the vehicle group, the animals treated with an HD of TanIIA presented a tPA expression level approximately six times greater, and those treated with an
" Fig. 3 A). In contrast, LD approximately three times greater (l the PAI-1 mRNA expression levels in the drug-treated groups were significantly (p < 0.05) decreased compared with those found in the vehicle group, and there were no significant differ" Fig. 3 B). The analyences between these drug-treated groups (l sis of COX-2 expression revealed that the HD of TanIIA reduced the COX-2 expression level to 60 % of the levels recorded in the vehicle-treated animals (p < 0.05). Similarly, the LD of TanIIA reduced the level of COX-2 mRNA to 82 % of that found in the vehicle-treated animals (p < 0.05). Altogether, TanIIA strongly " Fig. 3 B). downregulates COX-2 expression (l
Discussion !
TPA plays an important role in the peritoneal fibrinolytic system. Studies in humans have shown that decreased tPA activity or the overexpression of PAI-1 leads to more severe adhesions [16]. Sev-
Table 1 Intra-abdominal adhesion scores of groups.
a
Adhesion score
DEX
High dose of TanIIA
Low dose of TanIIA
Vehicle
0 1 2 3 4 Mean ± SD
6* 4 4 1 0 1.00 ± 1.00a, b
5 6 3 1 0 1.00 ± 0.93a, b
2 3 5 4 1 1.93 ± 1.16a
1 1 2 6 6 3.13 ± 0.92b
1 2 3 4 5
P < 0.05 compared with the vehicle group; bp < 0.05 compared with the LD of TanIIA group; * number of animals
Wang C et al. Prevention of Experimental …
Planta Med 2014; 80: 969–973
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Fig. 1 Chemical structure of TanIIA.
Fig. 3 TpA (A), PAI-1, and COX-2 (B) mRNA expression in peritoneal tissues. Relative expression levels of tPA, PAI-1, and COX-2 mRNAs are normalised against β-actin. Results are expressed as a percent of vehicles and shown as mean ± SEM (n = 5). * P < 0.05 compared with the vehicle, # p < 0.05 compared with the LD of TanIIA (2.5 mg/kg).
eral chemicals, such as NK-1RA, statins, octreotide, and methylene blue, have been proven to be effective for the prevention of PPA formation by promoting tPA activity and upregulating peritoneal fibrinolysis [17–19]. In our experiments, it was found that on postoperative day 7, the tPA activity level in the peritoneal lavage fluid was increased significantly in drug-treated groups. This suggested that TanIIA could increase the activity of the peritoneal fibrinolytic system and effectively decrease adhesion formation in the abdomen. Furthermore, we also found that the mRNA ex-
pression level of tPA increased in the parietal peritoneal tissue of the TanIIA-treated animals. In contrast, TanIIA significantly de" Fig. 4 B). The PAI-1 procreased the PAI mRNA expression level (l tein, which appears to be an acute-phase reactant protein [20], is the most important inhibitor of tPA and uPA [21, 22]. Segura et al. demonstrated that the RNA interference-mediated downregulation of the PAI-1 gene in rats can prevent abdominal adhesion formation [23]. The qPCR results discussed in this study correlate with the observed tPA activation in the peritoneal lavage fluid, which indicates that the upregulation of tPA mRNA expression and the downregulation of PAI mRNA can evoke tPA activation in the peritoneal cavity. Overall, these changes result in the upregulation of the fibrinolytic system. In addition to the PAI mRNA expression level, we found that the COX-2 mRNA expression level was also significantly downregulated both in TanIIA-treated and DEX-treated animals compared with the vehicle-treated animals. It has been confirmed that anti-inflammatory drugs, including NSAIDs such as aspirin, can decrease adhesion formation in animals [24]. Anti-inflammatory agents have been used to reduce the initial inflammatory response to tissue injury [25]. As one of the most important proinflammatory mediators, COX-2 has been found to induce proinflammatory, proangiogenic, and profibroblastic effects, all of which may promote adhesion formation. Inhibition of the COX-2 enzyme, which is expressed by adhesion fibroblasts [26], has been shown to reduce adhesion formation in rodents [27–29]. The marked reduction of COX-2 mRNA expression by TanIIA suggests a second mechanism for the prevention of adhesion formation after abdominal surgery. It has been found that TanIIA, by decreasing COX-2 expression induced by lipopolysaccharides, inhibits prostaglandin E2 synthesis in osteoblasts, which, in turn, suppresses inflammatory bone loss [30]. Two recent studies have shown that TanIIA has a protection effect on the cells which are located in ischaemia areas, such as in cases of myocardial ischaemia and cerebral ischaemia in rats [31, 32]. Another study has demonstrated that the cardioprotective effects of TanIIA might be attributed to its capacity for inhibiting inflammatory responses. In an in vitro study, TanIIA was found to attenuate the myocardial infarction pathological changes, improve heart function, and reduce the expression of MCP-1, TGF-β1, and macrophages in filtration [12]. It may be concluded that there is a possibility that TanIIA be applied to PPA diseases because it significantly inhibits PPA, mainly through the decreasing inflammatory response process and activating the fibrinolysis system. Further research needs to be carried out on plasma-derived mediators such as C3, plasmin, and factor XII as well as cell-derived mediators such as histamine, IFN, and IL so as to clarify the mechanism of TanIIA in reducing inflammatory response.
Fig. 4 A Creation of the ischaemic buttons to the peritoneal sidewall as previously described. B Adhesions to the buttons at postoperative day 7. (Color figure available online only.)
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Original Papers
Original Papers
Materials and Methods
Table 2 Grading of adhesions in rats according to the criteria of Nair [41].
!
Animal model
Grade
Description
Classification
Male Wistar rats weighing 175 to 200 g, which were obtained from the Laboratory Animal Centre of Southern Medical University (Guangzhou, China), were used for all of the experiments. The rats were housed at a constant room temperature with 12-h light/12-h dark cycles and were provided with standard rodent chow and water ad libitum. All of the procedures and animal care protocols were approved by the Committee on the Ethics of Animal Experiments of Southern Medical University (permit number: 44 020 101 922, date: 04/22/2012). The abdominal hair of the rats was shaved and the animals were anaesthetised through the administration of 5% pentobarbital sodium by injection into the right side of the abdomen. After the lower abdomen was shaved and cleansed with 75% alcohol, a 3to 4-cm midline laparotomy was performed. Four ischaemic buttons, spaced 1 cm apart, were created on both sides of the parietal peritoneum by grasping 5 mm of the peritoneum with a haemostat and ligating the base of the segment with a 4–0 silk suture. The cecum was then isolated and scratched with a scalpel [33]. All of the surgical procedures were performed under sterile conditions. As the DEX has long been proven as an effective reference drug in preventing PPA [34–39], it was administrated to the control group as a reference drug. Sixty rats were divided into four equal groups (n = 15 per group). Each of the groups was administrated DEX (10 mg/kg, 99 % purity, Shanghai Yansheng Industrial Co., Ltd.), vehicle (100 % DMSO), and different amounts of TanIIA (98% purity, Xiʼan Honson Biotechnology Co., Ltd.), which were categorised as HD (10 mg/kg) and LD (2.5 mg/kg). In all groups, the intraperitoneal administration was performed at the time of laparotomy [40].
0
Complete absence of adhesions
Insubstantial adhesions
1
Single band of adhesions between viscera or from one viscus to abdominal wall Two bands, either between viscera or from viscera to the abdominal wall More than two bands between viscera or viscera to the abdomimal wall, or whole of intestines forming a mass without being adherent to the abdominal wall Viscera directly adherent to the abdominal wall, irrespective of number and extent of adhesive bands
Grading of adhesions in rats On postoperative day 7, an abdominal incision was made using the previously described anaesthetic method. The adhesions " Fig. 4) were graded as described by Nair et al. [41], and the ad(l hesion scores were determined by an investigator blind to the di" Table 2). vision of the groups (l
The tissue-type plasminogen activator activity in the peritoneal lavage fluid The peritoneal fluid was collected by rinsing the peritoneal cavity with 2 mL of phosphate buffer containing heparin (1 IU/mL, pH = 7.4) at 37 °C. Approximately 1 mL of the fluid was recovered and added to an equal volume of 0.2 M sodium acetate, pH 3.9. The cellular debris was then removed by microcentrifugation (2000 rpm × 1 min, 4 °C) [42]. The fibrinolytic activity in each sample due to tPA was assayed in duplicate by adding 50 µL of the diluted sample to different wells of a 96-well microtitre plate containing 50 µL of tPA stimulator (0.6 mg/mL cyanogen bromide-digested fibrinogen, American Diagnostica, Inc.). Then, 150 µL of assay buffer [16.7 µg/mL human plasminogen (Athens Research and Technologies), 667 µM S-2251 substrate (Chromogenix), and 20 mM Tris, pH 8.3] was added to each well and gently mixed. Cleavage of the S-2251 substrate by tPA-activated plasmin produced a yellow colour that absorbs wavelengths of 405 and 490 nm. The change in absorbance was measured at 37 °C over a 6-h period using a Spectra Max 250 spectrophotometer. The activity of tPA in each sample was determined by extrapolation from a tPA (human; Prospec) standard curve [42].
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Planta Med 2014; 80: 969–973
2 3
4
Substantial adhesions
Detection of tissue-type plasminogen activator, plasminogen activator inhibitor, and cyclooxygenase-2 messenger ribonucleic acid expression levels by quantitative real-time polymerase chain reaction analysis The ischaemic button tissue of the parietal peritoneum, including a 1-mm rim of the surrounding tissue, was removed, frozen in liquid nitrogen, and stored at − 80 °C until qRT‑PCR analysis. The rats were then euthanised by a combination of pneumothorax and cardiac puncture. The ischaemic button tissues were homogenised by pulverisation in liquid nitrogen, and 1 mL of 0.1% Triton X 100 was added for every 40 mg of tissue. The total RNA from the peritoneal ischaemic button tissue was isolated according to the manufacturerʼs instructions (RNAiso Plus, Takara Biotechnology). The RNA concentrations were determined spectrophotometrically by measuring the absorbances at 260 and 280 nm, and the RNA quality was assessed through gel electrophoresis. The complementary DNA (cDNA) was synthesised from the total RNA using the All-inone™ first-strand cDNA synthesis kit (Genecopoeia), according to the manufacturerʼs suggested protocol, and qRT‑PCR was conducted using the All-in-one™ qPCR mix system (Genecopoeia) with SYBR green, according to the standard protocol. The following primer sets were used to amplify the following probes: " tPA, (F) 5′-CGCTGTACCTCACAGCATCTGTTTA‑3′ and (R) 5′-CATCCGCTTATCGATCATGCAC‑3′; " PAI-1, (F) 5′-ACCATCTCCGTGCCCATGA‑3′ and (R) 5′-GGGCAGTTCCAGGATGTCGTA‑3′; " COX-2, (F) 5′-AACACGGACTTGCTCACTTTGTTG‑3′ and (R) 5′-AATGGAGGCCTTTGCCACTG‑3′; " β-actin, (F) 5′-GGAGATTACTGCCCTGGCTCCTA‑3′ and (R) 5′-GACTCATCGTACTCCTGCTTGCTG‑3′. qRT‑PCR was performed using an Agilent Stratagene 3005P system. All PCRs were performed under the conditions recommended by the manufacturer: 95 °C for 10 min for the initial denaturation, 40 cycles of 95 °C for denaturation, 60 °C for 20 s for annealing, and 72 °C for 20 s for extension.
Statistical analysis The SPSS for Windows 10.0 software was used for the statistical analyses. The Kruskal-Wallis test was used to identify the differences in the adhesion grades between the groups. In addition, the Mann-Whitney U test was used to determine the differences be-
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tween each pair of groups. One-way ANOVA was performed to assess all of the other differences between the groups. If the differences were significant (p < 0.05), the Student-Newman-Keuls test was conducted to detect the differences between specific means. The results were evaluated at a significance level of p < 0.05.
Conflict of Interest !
The authors declare no conflict of interest.
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