Intraperitoneal Lavage with Crocus sativus Prevents Postoperative-Induced Peritoneal Adhesion in a Rat Model: Evidence from Animal and Cellular Studies

Postoperative peritoneal adhesions are considered the major complication following abdominal surgeries. The primary clinical complications of peritoneal adhesion are intestinal obstruction, infertility, pelvic pain, and postoperative mortality. In this study, regarding the anti-inflammatory and antioxidant activities of Crocus sativus, we aimed to evaluate the effects of Crocus sativus on the prevention of postsurgical-induced peritoneal adhesion. Male Wistar-Albino rats were used to investigate the preventive effects of C. sativus extract (0.5%, 0.25% and 0.125% w/v) against postsurgical-induced peritoneal adhesion compared to pirfenidone (PFD, 7.5% w/v). We also investigated the protective effects of PFD (100 μg/ml) and C. sativus extract (100, 200, and 400 μg/ml) in TGF-β1-induced fibrotic macrophage polarization. The levels of cell proliferation and oxidative, antioxidative, inflammatory and anti-inflammatory, fibrosis, and angiogenesis biomarkers were evaluated both in vivo and in vitro models. C. sativus extract ameliorates postoperational-induced peritoneal adhesion development by attenuating oxidative stress [malondialdehyde (MDA)]; inflammatory mediators [interleukin- (IL-) 6, tumour necrosis factor- (TNF-) α, and prostaglandin E2 (PGE2)]; fibrosis [transforming growth factor- (TGF-) β1, IL-4, and plasminogen activator inhibitor (PAI)]; and angiogenesis [vascular endothelial growth factor (VEGF)] markers, while propagating antioxidant [glutathione (GSH)], anti-inflammatory (IL-10), and fibrinolytic [tissue plasminogen activator (tPA)] markers and tPA/PAI ratio. In a cellular model, we revealed that the extract, without any toxicity, regulated the levels of cell proliferation and inflammatory (TNF-α), angiogenesis (VEGF), anti-inflammatory (IL-10), M1 [inducible nitric oxide synthase (iNOS)] and M2 [arginase-1 (Arg 1)] biomarkers, and iNOS/Arg-1 ratio towards antifibrotic M1 phenotype of macrophage, in a concentration-dependent manner. Taken together, the current study indicated that C. sativus reduces peritoneal adhesion formation by modulating the macrophage polarization from M2 towards M1 cells.


Introduction
Postoperative peritoneal adhesions are considered the major complication after abdominal surgery. Peritoneal adhesion is an abnormal connective tissue that occurs between two tissues that have been damaged during the surgery [1,2]. The peritoneum gets harmed and forms a temporary matrix during the surgery. After several hours, this provisional matrix becomes a clot, which can be destroyed by various factors such as macrophages and fibrinolysin enzymes.
Following the clot formation after 72 hours, the fibroblasts of the underlying tissues migrate into the clot and provide a field for forming sticky tissue [3,4]. It has been emphasised that inflammation, free radicals, hypoxia, coagulation, and fibrinolysis are the main pathophysiological reasons responsible for forming peritoneal adhesion [2,5].
The surgical technique is the first method for adhesion treatment; however, it is insufficient alone [12]. Other therapeutic approaches have been studied, such as barrier therapy [13,14] and gene therapy [15]. However, there is still no approved method for the treatment or prevention of adhesion, although a high prevalence of postoperative adhesions.
To our knowledge, there is no study evaluating the protective effects of C. sativus extract on preventing postoperative intra-abdominal adhesions. Therefore, in the present study, we aimed to determine the anti-inflammatory and antioxidant effects of Crocus sativus on the formation and prevention of postoperative abdominal adhesions in a rat model of peritoneal adhesion.

High-Performance Liquid Chromatography-(HPLC-) Mass Spectrometry (MS) Apparatus and the Extracted
Analysis. The LC-MS analysis was performed in an AB SCIEX QTRAP (Shimadzu) liquid chromatography coupled with a triple quadrupole Mass Spectrometer. Liquid chromatography separation was performed on a Supelco C18 (15 mm × 2:1 mm × 3 μm) column. MS analysis was carried out in both negative and positive modes of ionisation to monitor as many ions as possible and to ensure that the most significant number of metabolites extracted from the C. sativus sample was detected. The analysis was done at a flow rate of 0.2 ml/min. The gradient analysis started with 100% of 0.4% aqueous formic acid, isocratic conditions were maintained for 1 min, and then a 14 min linear gradient to 40% acetonitrile with 0.4% formic acid was applied. From 14 to 35 min, the acidified acetonitrile was increased to 100%, followed by 5 min of 100% acidified acetonitrile and 5 min at the start conditions to reequilibrate the column. The mass spectra were acquired in a range of 100 to 1500 within the 45 minutes scan time. Mass feature extraction of the acquired LC-MS data and maximum detection of peaks was done using the MZmine analysis software package, version 2.3.

In Vivo Study
2.4.1. Animals. Seventy male Wistar-Albino rats weighing 250 ± 15 g (six weeks old) were purchased from the animal laboratory unit of Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran. Rats were housed in separated standard cages and ventilated room with a 12/12 h natural light-dark cycle, 60 ± 3% humidity, and temperature of 21 ± 2°C. They had free access to food and taped water before and during the experiments. More appropriate hygiene was provided with continuous cleaning and removal 2 Oxidative Medicine and Cellular Longevity of faeces and spilt feeds from cages daily. All animals received human care in compliance with institutional guidelines.  [2,24,25]. In summary, animals received 100 mg/kg of ketamine and 10 mg/kg of xylazine intraperitoneally (i.p.) for anaesthesia. Following the skin's shaving and disinfection with alcohol and iodine solution, a three-centimeter incision was carefully done to reach the abdominal cavity. For intra-abdominal adhesion induction in rats, the peritoneal abrasion method was performed as one side of the middle abdominal incision was gently abraded using a soft sterilised paper polisher until the cecum provided an opaque presentation with fine petechiae. Afterwards, the peritoneum and the injured area were washed by 2 ml of the extract or vehicle. After the intervention, the cecum was returned to the abdomen and abdomen wall then closed with 4-0 poly-gelatine suture. The procedure lasted to a maximum of 10 minutes. After surgery, rats were kept in their cages in the recovery room for seven days. All treatments were done by lavage in the abraded and whole surgical zone with a 2 ml syringe. Furthermore, all rats received a single dose of antibiotic cefazolin (300 mg/kg intramuscularly; i.m.) immediately after ending the surgery to prevent possible wound infection [26][27][28].

Experimental Groups.
Seventy male Wistar rats were randomly divided into seven groups containing ten animals and grouped as follows: (1) Group 1: normal-rats received neither surgical nor intervention procedures.
(2) Group 2: control-rats received surgical and peritoneal adhesion procedures without treatment.
(3) Group 3: vehicle-rats received surgical and peritoneal adhesion procedures and were treated with 2 ml of the vehicle (the vehicle was sterilised distilled water containing 5% v/v of tween 80 [2]).

Assessment of the Macroscopic Adhesion Grade.
On the seventh day after the surgery, rats underwent a second laparotomy. Thereafter, two independent researchers blind to the protocol assessed the adhesion grading using the score published by Nair et al. [32] (Table 1). Additionally, cecum and peritoneal lavage fluid were collected for the measurement of inflammatory, fibrotic, and oxidative biomarkers.

Histological
Assessment. In the current experiment, paraffin-embedded histological sections were stained by Masson's trichrome staining to assess the extent and distribution of fibrosis in rats' peritoneal tissue as described in previous studies [33][34][35]. In this regard, after removing formalin and washing with distilled water three times, the tissues were transferred to different alcohol concentrations (50-100%) for some minutes. Tissue sections were observed with magnifications of 4x, 20x, and 40x using a Nikon E-1000 microscope (Japan) under bright-field optics.
2.4.6. Evaluation of Oxidative Parameters. The levels of MDA, as an oxidative marker, and GSH, as an antioxidative marker, were measured in the peritoneal fluid using biochemistry kits (ZellBio®, Germany) according to the manufacturer's manuals [36,37].

Assessment of Inflammatory and Anti-Inflammatory
Biomarkers. The levels of TNF-α, IL-6, and PGE 2 , as inflammatory markers, and IL-4 and IL-10, as anti-inflammatory markers, were evaluated in peritoneal lavage fluid by ELISA kits (Bender Med®, Germany) according to the manufacturer's instruction [38,39].

Evaluation of Fibrosis and Angiogenesis Biomarkers
and Tissue Plasminogen Activator (tPA) and Plasminogen Activator Inhibitor (PAI). According to the manufacturer's instruction, the concentrations of fibrosis biomarkers (TGF-β) and angiogenesis marker (VEGF) of peritoneal fluid specimens were assessed by the relevant ELISA kits. Two bands, either between viscera or from viscera to the abdominal wall 3 More than two bands, between viscera or viscera to the abdominal wall or whole intestines forming a mass without being adherent to the abdominal wall 4 Viscera directly adherent to the abdominal wall, irrespective of number and extent of adhesive bands 3 Oxidative Medicine and Cellular Longevity Additionally, according to the manufacturer's instruction, the levels of tPA, which digests fibrin substrates, and PAI were also evaluated in peritoneal lavage fluid by ELISA kits. Subsequently, the tPA/PAI ratio was calculated by dividing the level of tPA by PAI level. The levels of cytokines were reported as pg/mg protein.

Proliferation Assay.
To investigate that C. sativus extract had no cytotoxicity and inhibitory effects on RAW 264.7 cells, the cells were cultured at a density of 3 × 10 3 cells/well in 96-flat well plates and incubated overnight [40]. Thereafter, the cells were incubated with different

Assessment of Secretory Cytokines Levels and
Intracellular Levels of iNOS and Arg-1. According to the manufacturer's instructions, the anti-inflammatory (IL-10) levels and inflammatory cytokine (TNF-α) and angiogenesis factor (VEGF) were measured by the ELISA-based method. The cells were cultured in 6-well plates (2 × 10 6 cells/each well) and incubated with different concentrations of C. sativus extract (100, 200, and 400 μg/ml, according to the preliminary evaluation), PFD (100 μg/ml, as a positive control group, [41]), or vehicle (contained 0.1% dimethyl sulfoxide, DMSO) in the presence of recombinant mouse TGF-β1 stimulation (20 ng/ml, providing M2 phenotype cells [42]) for 24 h and then coincubated with TGF-β1 (20 ng/ml [42]) for another 24 h, at 37°C in a 5% v/v CO 2 incubator. Finally, the supernatants were collected to measure the levels of cytokines. The levels of cytokines were reported as pg/mg protein. Moreover, the cells were collected and lysed using a lysis buffer and then homogenised (DIAX 100, Heidolph, Schwabach, Germany) on the cold water (0-4°C) for 2-3 min along with vortexing (every 30 sec). The samples were centrifuged at 12,000 g for 10 min at 4°C, and 50 μl of supernatants had then undergone an assessment. The levels of iNOS and Arg-1 were reported as ng/mg protein.
2.6. Statistical Analysis. Data were analysed using GraphPad Prism (version 6.01) software and presented according to the nature of parametric or nonparametric as the means ± SEM or median ± interquartile range, respectively. P values ≤ 0.001, 0.01, and 0.05 were statistically considered significant. For parametric data, one-way ANOVA was performed     9 Oxidative Medicine and Cellular Longevity with the following Tukey's Kramer post hoc test. However, for nonparametric data (adhesion score), the Kruskal-Wallis test was done following Dunn's multiple comparisons posttest. The data and statistical analysis comply with the recommendations on experimental design, analysis [44], and data sharing and preclinical pharmacology presentation [45,46].  Figures 1(a) and 1(b), respectively. The MS spectral data were compared with the reported compounds in some previous literature. Figures 1(a)-1(f) are examples of extracted ion chromatograms from the total ion chromatogram and its related mass. Some flavonoids, including quercetin 3-oru-tinosylrhamnoside, quercetin 3-O-rutinoside, Kaempferol 3glucoside, tamarixetin 3-O-bihexoside, rhamnetin, and naringenin, were detected in C. sativus L. extract. Apocarotenoids, including crocin, crocetin, and their derivatives, apart from imparting colours to C. sativus, also have antioxidant properties (40).

In Vivo Results
3.2.1. The Effect of C. sativus and PFD on Adhesion Score. The adhesion scores in both the control and vehicle groups were increased compared to those in the normal group (P < 0:001 for both cases, Figures 2(a)-2(c)). Treatment with PFD (7.5% w/v, P < 0:01) and C. sativus (0.25% w/v, P < 0:01, and 0.5% w/v, P < 0:001) significantly attenuated the levels of adhesion score compared to the control group (Figure 2(a)). The frequencies of adhesion score are indicated in Figure 2  results showed the levels of tissue fibrosis and collagen deposition (blue colour) in both the vehicle and control groups (Figure 2(d)). On the contrary, the blue colour's intensities were notably lower in all doses of the extract groups and PFD as a positive control than the control group (Figure 2(d)).

The Effect of C. sativus and PFD on MDA and GSH.
The concentrations of MDA (P < 0:001, Figure 7(a)) and GSH (P < 0:001, Figure 7(b)) were significantly increased and decreased in the control group compared to the normal group, respectively. The levels of MDA and GSH, respectively, diminished and increased following treatment with C. sativus (0.25, 0.5%w/v) and PFD (7.5%w/v) in comparison to the control group in peritoneal lavage fluid (P < 0:001 for all cases, Figures 7(a) and 7(b)).

The Effect of C. sativus Extract and PFD on Cell
Proliferation. In the absence of TGF-β 1 stimulation, no significant changes were found in cell proliferation between the groups treated with vehicle, C. sativus extract (100, 200, and 400 μg/ml) and PFD (100 μg/ml) and the control group (Figure 8(a)). In the presence of TGF-β 1 stimulation (20 ng/ml), the levels of cell proliferation were significantly increased in both vehicle-treated and TGF-β 1 groups compared to the respected control group (P < 0:001 for both cases, Figure 8 13 Oxidative Medicine and Cellular Longevity decreased the level of cell proliferation compared to the TGF-β1-treated alone group (P < 0:001 for all cases, Figure 8(b)). The potential protective effects of C. sativus extract (100 and 200 μg/ml) were lower than those of PFD (100 μg/ml) on decreasing the TGF-β 1 -induced cell hyperproliferation (P < 0:001 for both case, Figure 8(b)).

Discussion
The present study evaluated the protective effects of hydroethanolic extract of C. sativus stigma against postoperational-induced peritoneal adhesion in a rat model. As a result, the current study demonstrated that C. sativus extract ameliorates postoperational-induced peritoneal adhesion development through attenuating oxidative stress (MDA), inflammatory mediators (IL-6, TNF-α, and PGE 2 ), and fibrosis (TGF-β1, IL-4, and PAI) and angiogenesis (VEGF) markers, while propagating antioxidant (GSH), anti-inflammatory (IL-10), and fibrinolytic (tPA) markers and tPA/PAI ratio. Moreover, we assessed the protective and antifibrotic effects of the extract against TGF-β1-induced fibrosis in RAW 264.7 murine macrophage cell line. Briefly, we revealed that the extract, without any toxicity, modulated the levels of cell proliferation and inflammatory (TNF-α), angiogenesis (VEGF), anti-inflammatory (IL-10), M1 (iNOS), and M2 (Arg-1) biomarkers and iNOS/Arg-1 ratio towards antifibrotic M1 phenotype of macrophage, in a concentration-dependent manner. Numerous models have been suggested to evaluate postoperative peritoneal adhesion, including uterine horn damage, bacterial infection, and scarping model [47,48]. In the current study, we used the scraping model due to the most similarity between the adhesion development by this model and abdominopelvic surgery [49,50]. Furthermore, we scored the adhesions from zero to four using the Nair et al. and adhesion scheme scoring methods [25,32,50]. Our macroscopic data revealed that the adhesion score was Data were presented as the mean ± SEM (n = 6). +++ P < 0:001, compared with the control group; * * * P < 0:001 and * P < 0:05 compared with the TGF-β 1 group; ### P < 0:001 to # P < 0:05 compared with the PFD group. PFD was increased TNF-α level, but it had no significant difference compared to the TGF-β 1 group (P = 0:0729).
significantly increased in the control group, while C. sativus (0.25 and 0.5% w/v) concentration-dependently reduced the adhesion formation following postoperational-induced peritoneal adhesion in the rat. Our previous study also reported that the adhesion score is enhanced in the control group that received postoperative-induced peritoneal adhesion and decreased following the interventions, such as propolis, honey, and Rosmarinus officinalis treatments [2,24,25,32].
In the present study, we used pirfenidone (PFD), a wellknown antifibrotic medicine, as a positive control. We showed that PFD (7.5% w/v) provided a significant decrement in adhesion score, MDA, TNF-α, PGE 2 , IL-6, IL-4, TGF-β, VEGF, and PAI levels, while making a significant increment in GSH, IL-10, and tPA levels as well as tPA/ PAI ratio following postoperational-induced adhesion in the rat. Moreover, following the TGF-β1 stimulation, our cellular results also revealed that PFD (100 μg/ml) significantly reduced the levels of cell proliferation, VEGF, and Arg-1 but notably enhanced IL-10, iNOS, and iNOS/Arg-1 ratio (M1/M2 marker) and polarized the macrophage from fibrotic phenotype towards antifibrotic M1 cells. Following our results, Bayhan et al. indicated that oral administration of PFD (500 mg/kg po~6.25% w/v) for two weeks significantly reduced adhesions grade and the protein concentrations and mRNA expression levels of matrix metallopeptidase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), tumour necrosis factoralpha (TNF-α), and TGF-β1 [29]. Similarly, Ozbilgin and coworkers reported the protective effects of PFD (150 mg/ animal~2 ml of 7.5% w/v) against peritoneal adhesion. In fact, they showed that PFD as the same concentration which used in our study (2 ml of 7.5% w/v) significantly diminished the peritoneal adhesion by decreasing the Th2 lymphocytes as fibrotic cells and increasing the Th1 lymphocytes as antifibrotic cells [31]. Moreover, in 2016, Hasdemir et al. also supported that intraperitoneal administration of PFD (150 mg/ animal ip~2 ml of 7.5% w/v) significantly abolished adhesion scores, fibrosis, and vascular proliferation as well as the protein concentrations of IL-17 and TGF-β1 [30]. Intriguingly, in the cellular model of adhesion, PFD at 100 μg/ml reprogrammed the IL-4/IL-13-induced M2 fibrotic macrophages and polarized towards M1 cells by decreasing the levels of TGF-β1, collagen type one, and related markers, including YM-1 and CD206 and transferrin receptors [41]. Collectively, these studies can support the results of the positive control PFD used in the current study. Data were presented as the mean ± SEM (n = 6). +++ P < 0:001, compared with the control group; * * * P < 0:001 and * P < 0:05 compared with the TGFβ 1 group; ### P < 0:001 and ## P < 0:01 compared with the PFD group. 16 Oxidative Medicine and Cellular Longevity It has been demonstrated that oxidative stress is one of the major factors responsible for adhesion development. Activated oxygen and nitrogen species stimulate fibroblastic cells' growth in damaged areas and lead to fibrosis formation [51,52]. Therefore, we investigated MDA levels as an oxidative agent and GSH as antioxidative factors. We found that C. sativus extract (0.25-0.5% w/v~25 and 50 mg/kg) meaningfully reduces MDA level and enhances GSH level following postoperational-induced peritoneal adhesion in a concentration-dependent manner. In line with our results, Ghadrdoost et al. determined that C. sativus extract (30 mg/kg) and crocin (15 and 30 mg/kg) diminish lipid peroxidation by reducing the MDA level. Simultaneously, the extract and its active constituent augmented total antioxidant activity, glutathione peroxidase, glutathione reductase, and superoxide dismutase activities following the oxidative stress and spatial learning and memory deficits induced by chronic stress in rats [53].
Additionally, it has been demonstrated that C. sativus aqueous extract (10, 20, and 40 mg/kg) mitigated MDA and nitric oxide levels, while it appended the levels of GSH and catalase and SOD activities following streptozotocininduced diabetes in rats [54]. Akbari and coworkers figured out that C. sativus extract (40 mg/kg) attenuates MDA and IL-6 levels and propagates GSH level as well as glutathione peroxidase activity in exercised rats [55]. In one study, C. sativus stigmas and high-quality byproducts (petals +anthers-CTA) extracts (25 μg/ml) provided a significant decrement in ROS and lactate dehydrogenase levels in human colon cancer (HCT116) cell lines following hydrogen peroxide-induced oxidative stress. Moreover, CST and CTA alleviated MDA levels in rat colon specimens challenged with E. coli lipopolysaccharide [56]. Crocin, one of the major active constituents of C. sativus, decreased MDA and xanthine oxidase while it increased GSH levels in streptozotocin-induced diabetic rats [57]. These studies may endorse our results regarding the antioxidant effects of C. sativus extract.
Inflammation and inflammatory cytokines are considered one of the most critical factors responsible for postoperative adhesion formation. In damaged tissue, macrophages secret IL-6 and TNF-α, which cause coagulation and the formation of fibrin layers that extend adhesion [3]. By contrast, IL-10 as an anti-inflammatory cytokine inhibits the secretion of pro-inflammatory cytokines, such as IL-8, IL-6, and TNF-α, and plasminogen activator enzymes and prevents tissue damage [53]. Therefore, we measured the effects of C. sativus on the levels of TNF-α, IL-6, IFN-γ, and PGE 2 as inflammatory cytokines and IL-4 and IL-10 concentrations as anti-inflammatory cytokines. Our results revealed that C. sativus extract (0.25-0.5% w/v) concentration-dependently reduces the levels of TNF-α, IFN-γ, PGE 2 , IL-6, and IL-4, while making a significant increment in IL-10 level following postoperational-induced adhesion in the rat. In line with our animal results, we observed that the level of IL-10 was increased following the TGF-β1 stimulation in the macrophage cell line. However, the level of TNF-α as an inflammatory cytokine was propagated at higher concentrations of the extract. In fact, this phenomenon was in contrast to the anti-inflammatory effects of the C. sativus extract observed in the animal section. It can be justified that TGF-β1 slightly reduces the TNF-α and leads to provide fibrotic macrophages (M2 cells), which produce higher levels of fibrotic and angiogenesis factors, as shown in our results of Figures 9 and 10. Indeed, by TGF-β1 stimulation, the macrophage phenotypes were polarized towards M2 cells by decreasing the level of increasing the level of Arg-1 as a marker of M2 cells and iNOS as a marker of M1 macrophage cells and iNOS/Arg-1 ratio (M1/M2 ratio). It justifies that the extract provides no inflammatory state but modulates the macrophage polarization towards nonfibrotic phenotypes that secrets higher TNF-α levels. Moreover, we assessed the level of IL-10 as supportive data, which endorse our vision on the direct effects of the extract on macrophage polarization and increasing the TNF-α level.
The previous human and animal studies indicated that the levels of TGF-β are significantly increased in the peritoneal adhesions [2,24,50]. TGF-β is a suppressive and fibrotic cytokine that controls reproduction, differentiation, cell apoptosis, and wound healing. The active form of TGF-β increases the secretion of the extracellular matrix, leading to the formation of adhesion [3,63]. Vascular endothelial growth factor (VEGF) is another growth factor and potent mitogen for endothelial cells and a vital angiogenesis factor, which is essential for wound healing and adhesion formation [2,24,50]. In fact, VEGF production is stimulated by lactate in macrophages, and lactate accumulation plays a critical role in adhesion development [2,3,24,50]. It has been emphasised that the anti-VEGF monoclonal antibody decreases the postoperational peritoneal adhesion in mice [64]. The current study results figured out that C. sativus extract (0.25-0.5% w/v~25 and 50 mg/kg) provided a significant and concentration-dependent decrement in TGFβ and VEGF levels following the postoperational peritoneal adhesion. Interestingly, our in vitro study found that VEGF level was also meaningfully abrogated by C. sativus extract in a concentration-dependent manner.
Tissue plasminogen activator (tPA) is classified as a serine protease that prevents the progression of mesothelial cell adhesion by inhibiting plasminogen transformation to plasmin. In low tPA level condition, fibrin masses form a clot attacked by fibroblasts, collagens, and other proteins that lead to adhesion formation [72]. Plasminogen activator inhibitor (PAI), which is present in plasma, inhibits the tPA. Increasing the PAI level and decreasing the tPA level and tPA/PAI ratio are considered adhesion development causes [3,73]. In one study, Atta and coworkers found lower TGF-β1 and PAI and higher tPA levels in the group with a lower rate of postoperative adhesion formation in rats [74]. Therefore, we determined the levels of TPA, PAI and the ratio of TPA/PAI. We found that C. sativus (0.25-0.5% w/ṽ 25 and 50 mg/kg) mitigates PAI level and propagates tPA and TPA/PAI ratio levels in a concentration-dependent manner following the postoperational induced peritoneal adhesion. Tsantarliotou and coworkers suggested that crocin at both low and high doses (10 and 100 mg/kg) could diminish PAI-1 levels in the liver and brain tissue following lipopolysaccharide-induced thrombosis in rats [75].

Conclusion
In summary, our results revealed that C. sativus could prevent postoperative peritoneal adhesion through attenuating adhesion score, oxidative stress, inflammatory cytokines, fibrosis, and angiogenesis markers, while propagating antioxidant and anti-inflammatory markers and tPA ( Figure 11). Moreover, the current study indicated that C. sativus reduces peritoneal adhesion formation by modulating the macrophage polarization from M2 towards M1 cells ( Figure 11). It could be concluded that C. sativus may be the right candidate for preventing postoperative peritoneal adhesion.

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Oxidative Medicine and Cellular Longevity