The Toll-like receptor 4 antagonist TAK-242 in combination with sodium hyaluronate alleviates postoperative abdominal adhesion in a mouse model

Abstract

Postoperative abdominal adhesion is one of the most common complications after abdominal surgery. The Toll-like receptor 4 (TLR4) signaling pathway is one of the most common inflammation-related pathways, and it has been demonstrated that TLR4 is highly expressed in adhesive tissues; however, the function of TLR4 in adhesion formation has not yet been studied. In the present study, the expression of TLR4 was first detected by immunohistochemical (IHC) and double-immunofluorescence staining in 40 mice, which were randomly divided into four groups, and sacrificed at 1, 3, 5 and 7 days after surgery. Subsequently, another 40 mice were randomly divided into five groups; with the exception of the sham group, the other groups were modeled and treated with saline that contained DMSO, sodium hyaluronate (HA), TAK-242 or TAK-242 + HA (applied to damaged peritoneal wounds). A total of 7 days after surgery, the mice were sacrificed and specimens were collected. Inflammation was detected by hematoxylin and eosin staining, and ELISA of transforming growth factor- β1 (TGF-β1) and interleukin-6 (IL-6); collagen deposition was examined by Masson staining and IHC staining of α-SMA; and reactive oxygen species (ROS) were detected by ROS staining and malondialdehyde (MDA) assay. The results revealed that TLR4 was highly expressed in the adhesive tissues at 3, 5 and 7 days after surgery. In addition, TAK-242 + HA treatment could reduce abdominal adhesion formation, exhibiting lower Nair’s score and inflammation scores, lower TGF-β1 and IL-6 levels, and lower collagen thickness and α-SMA levels compared with those in the control group. In addition, the TAK-242 + HA group had lower levels of ROS and MDA compared with those in the control group. The present study revealed that TLR4 was highly expressed in the process of adhesion formation and its inhibitor, TAK-242, combined with HA, could reduce adhesion formation by reducing inflammation and ROS, and alleviating collagen deposition.

Introduction

Abdominal adhesion is one of the most troublesome postoperative complications that arise after abdominal or pelvic surgery, which can result in a series of problems, including chronic abdominal pain, ileus and female infertility, and may necessitate reoperation [1, 2]. According to statistical analysis, the incidence rate of postoperative abdominal adhesions is up to 66% [3]. It is well known that postoperative abdominal adhesion formation is mainly due to the increased deposition of fibrin over fibrinolysis [4]. Currently, clinical therapy to alleviate abdominal adhesions includes physical, medical and surgical treatments [5, 6]; however, the clinical efficacy remains unsatisfactory and may bring undesirable complications and side effects. Notably, abdominal adhesions are associated with extra costs and health risks for patients and medical institutions [7, 8]. Although previous studies have aimed to identify preventative options, such as medications or natural and synthetic barrier materials, the effectiveness of these approaches remains unsatisfactory [5]. Thus, it is critical to discover practicable drugs and to determine the potential mechanisms of abdominal adhesions to alleviate the condition. Sodium hyaluronate (HA) is a typical antiadhesion agent that is generally used to prevent PAA, which have been shown to be effective in animal studies and have been used to prevent postoperative intra-abdominal adhesion in clinical practice [9, 10].

It has been demonstrated that inflammation plays an important role in the formation of postoperative abdominal adhesions [11, 12], as follows: After injury or trauma, increased vascular permeability leads to the release of a large number of inflammatory mediators and inflammatory cells, resulting in the exudation of an excess of fiber-rich substances into the damaged local peritoneal tissues; this initiates the formation of adhesive bands. If the exudated fluid is absorbed within 3–5 days, no adhesion will be formed, otherwise a firm adhesive band may be created [13, 14]. Therefore, it is necessary to inhibit the inflammatory response and its expansion to reduce the formation of postoperative abdominal adhesions.

Oxidative stress is defined as an imbalance between the production and the removal of reactive oxygen species (ROS) [15, 16]. Certain concentrations of ROS are beneficial in killing pathogenic microorganisms and promoting tissue healing; however, an excessive accumulation of ROS promotes inflammation and affects peritoneal mesothelial cell repair in adhesive tissues [11, 17]. Moreover, early alleviation of the damage caused by oxidative stress during the formation of abdominal adhesions can effectively prevent adhesions [18]. Our previous studies demonstrated that ROS inhibition can reduce adhesions [19, 20]. However, there is a lack of research on the key molecular mechanisms and possible targeted drugs involved in oxidative stress response in adhesion formation.

Toll-like receptor 4 (TLR4) is a pathogen-associated molecular pattern that plays a pivotal role in the activation of innate immunity [21, 22]. Various tumor cell lines have been proven to express TLRs, which can be directly activated by the aforementioned antigens and ligands [23]. This can result in the creation and persistence of such a chronically inflamed microenvironment promoting cell proliferation [24]. Numerous TLR4 inhibitors have been developed using a variety of scaffolds. To date, three TLR4 inhibitors have been used in clinical trials, which can significantly alleviate symptoms in septic shock [25]. TAK-242 represents a novel therapeutic approach to the treatment of TLR4-mediated diseases. In a study of liver ischemiareperfusion injury (IRI), mechanistic experiments revealed that TAK-242 pretreatment significantly inhibited the IRIassociated inflammatory response by alleviating mitochondrial dysfunction, and reducing ROS and malondialdehyde (MDA) levels [26]. A previous study demonstrated that TLR4 is highly expressed in adhesive tissues [27]. Furthermore, it has been reported that the effect of COX-2 on lipopolysaccharideinduced postoperative peritoneal adhesion is achieved by targeting TLR4 [28]. Nevertheless, the efficacy of TLR4 inhibition in preventing adhesion formation has not yet been investigated.

Consequently, it was hypothesized that targeting TLR4 may have considerable potential for the management of abdominal adhesions; however, further research is required to elucidate the precise mechanism of action of this approach. The present study detected the expression of TLR4 in adhesive tissues, and then explored whether applying the TLR4 inhibitor TAK-242 combined with sodium hyaluronate (HA) to damaged peritoneal wounds could effectively prevent the formation of postoperative abdominal adhesions in a mouse model. Furthermore, the levels of inflammation and oxidative stress were quantified in adhesive tissues. Taken together, the present research provides a novel and specific insight into preventing postoperative abdominal adhesions.

Materials and methods

Animals and generation of the postoperative abdominal adhesion model

C57BL/6 mice were maintained at 22 ± 2˚C, and were given free access to food and water. The experimental scheme was approved by the Bioethics Committee of the Department of Medicine, Xi’an Jiaotong University (approval no. 2024 − 1916). All methods used in the present study were carried out in accordance with relevant regulations and the ARRIVE guidelines. All mice, except for those in the sham group, underwent model surgery. The surgery was performed as follows: The mice were anesthetized with isoflurane until they were fully unconscious, as confirmed by the toe-pinch test. Then, the mice were sterilized and a 1-cm midline abdominal incision was made, the cecum was pulled out of the abdominal cavity and the abdominal wall of the right lower abdomen and the wall of the cecum were rubbed with dry gauze 30 times until needle-point bleeding sites appeared [29]. The injured cecum was then placed in the abdominal cavity opposite the injured abdominal wall. Finally, the abdominal wall was closed with 4.0 absorbable sutures. The mice were allowed free access to food and water 8 h after surgery.

Groups and treatments

A total of 40 male C57BL/6 mice aged 8–12 weeks were randomly divided into five groups. The mice in the sham group only underwent laparotomy, whereas the other groups underwent adhesion model surgery. Before closing the abdominal cavity, different drugs were applied to the damaged peritoneal wounds. The control group was treated with 1 ml saline (containing 0.9% DMSO), the TAK-242 group was treated with 1 ml TAK-242 [3 mg/kg; Selleck; first dissolved in DMSO and diluted with saline (the final DMSO concentration was 0.9%)] [30], the HA group was treated with 1 ml HA (Zhejiang Jingjia Medical Technology Co.) and the HA + TAK-242 group was treated with 1 ml HA + TAK-242 mixed gel (TAK-242 was first dissolved in DMSO and mixed with the HA gel). The mice were fasted for 4 h after surgery.

After euthanasia, the abdominal cavity of the mice was opened with a U-incision, and the adhesive tissues or normal peritoneal tissues were collected and divided into two sections: One fixed in 10% formalin and the other frozen at -80˚C, for subsequent experiments. Adhesion was graded by two uninformed researchers according to Nair’s score: Grade 0, no adhesion; Grade I, one adhesion zone between the viscera, or between the viscera and the abdominal wall; Grade II, two adhesion zones between the viscera, or between the viscera and the abdominal wall; Grade III, more than two adhesion zones, while the viscera did not adhere directly to the abdominal wall; Grade IV, viscera adhered directly to the abdominal wall, regardless of the number of adhesion zones.

In addition, to examine TLR4 expression in adhesive tissues, 40 mice were randomly divided into four groups, and the mice were subjected to abdominal adhesion surgery, and sacrificed at 1, 3, 5 and 7 days after surgery. The adhesive tissues were collected, and the normal cecum and abdominal wall peritoneal tissues were used as controls. The tissues were fixed in 10% formalin or stored at -80˚C for further experiments.

Hematoxylin and eosin (H&E) staining

Then five randomly selected samples per group were cut longitudinally, and the middle parts were fixed in 10% formalin, embedded in paraffin and sliced into 4 mm sections for follow-up experiments. The sections were first dewaxed with xylene and then stained with H&E solution (Wuhan Servicebio Biotechnology Co. Ltd.), as described previously. Inflammation was scored as follows: 0, tissues without inflammatory cells; 1, samples with observable macrophages, lymphocytes and plasma cells; 2, samples with macrophages, plasma cells, eosinophils, and neutrophils; and 3, samples with inflammatory cell infiltration and microabscess formation. For each section, at least five high-power fields were selected for scoring, and the process was performed by two independent investigators.

Sirius red staining and Masson staining

At least five pathological sections of each tissue were randomly selected for Sirius red staining and Masson staining, which were performed using kits from Wuhan Servicebio Biotechnology Co., according to the manufacturer’s instructions. At least 10 high-power magnification fields were selected for each section, and the thickness of collagen fibers in each field was measured by microscopy.

ELISA

For ELISA, fresh specimens were collected, 10% homogenates were prepared mechanically in an ice-cold water bath and were centrifuged at 2,500-3,000 rpm for 10 min, and the supernatant was taken for determination. The inflammatory markers interleukin-6 (IL-6) and transforming growth factor-β1 (TGF-β1) were measured using customized ELISA kits (Hangzhou Lianke Biotechnology Co., Ltd. for TGF-β1; Thermo Fisher Technologies for IL-6), according to the manufacturers’ instructions. Within 10 min of the end of the reaction, the OD value of each well was measured at 450 nm using an ELISA plate reader (BioTek Epoch, United States) after zeroing the blank control well for quantitative protein concentration.

Immunohistochemical (IHC) and immunofluorescence staining

IHC staining was performed using a kit purchased from Wuhan Servicebio Biotechnology Co., according to the manufacturer’s instructions. At least three sections of each tissue were randomly selected for IHC staining. After dewaxing, the sections were placed in 3% hydrogen peroxide solution and treated with 3% BSA (GC305010, Wuhan Servicebio Biotechnology Co. Ltd.). Subsequently, the sections were incubated with primary antibodies against TLR4 (GB12186, 1:500; Wuhan Sanying Biotechnology Co.) and α-SMA (GB13044, 1:500; Wuhan Sanying Biotechnology Co.) overnight, and, after washing, the sections were incubated with secondary antibodies (HRP-conjugated antibody or a fluorescent secondary antibody) in a humidified incubator at 37˚C for 30 min. Next, DAB was used for color development. Finally, the sections were observed under a microscope and images were captured. IHC scoring was performed as follows: Intensity was scored as 0 for no expression, 1 for weak expression, 2 for moderate expression and 3 for strong expression. The area of staining was scored as 0 if < 5% of the area was stained, 1 if 6–25% of the area was stained, 2 if 26–50% of the area was stained, 3 if 51–75% of the area was stained, and 4 if > 75% of the area was stained. The total score was determined by multiplying the intensity score by the positive area score. The IF staining score was calculated according to the formulae.

Measurements of ROS

Tissues stored at -80˚C were processed into serial frozen sections and at least five sections were used to detect ROS in the tissues. After the sections were slightly dehydrated, the ROS staining solution (Wuhan Servicebio Technology Co. Ltd.) was added to the sectioned tissues and incubated for 30 min at 37˚C in the dark. After being washed three times, the slices were incubated in DAPI solution (Wuhan Servicebio Technology Co. Ltd.) for 10 min at room temperature. Subsequently, the sections were washed three more times. Images were collected after the anti-fluorescence quenching tablet was sealed and these sections were observed by fluorescence microscopy and images were captured. ROS levels were calculated as the ratio of ROS-positive nuclei to total nuclei.

MDA assay

MDA was detected using a kit purchased from Beijing Solebro Reagent Co. Briefly, fresh specimens were mechanically homogenized and the supernatant was collected and mixed with the MDA assay working solution for determination. The mixture was dispensed and maintained at a temperature of 100˚C in a water bath for 60 min, before being cooled in an ice-cold water bath and centrifuged at room temperature for 10 min. A volume of 200 µl supernatant was then added to a 96-well plate. The absorbance of each sample was measured at an OD of 532 and 600 nm, and the MDA content was calculated according to the formulae.

Statistical analyses

Data collection and analyses were performed using SPSS 18.0 (SPSS Inc., USA). The results are presented as percentages, absolute numbers or as the mean ± standard deviation. Student’s t-test (two-tailed) or one-way ANOVA were used to analyze normally distributed data. The Kruskal-Wallis test was used to analyze non-normally distributed data. All the results were revised using Bonferroni’s correction. The χ² test or Fisher’s exact test were used to analyze qualitative data. P < 0.05 was considered to indicate a statistically significant difference.

Results

TLR4 is highly expressed in adhesive tissues

A previous bioinformatics analysis reported that TLR4 may be highly expressed in adhesive tissues, which was verified by animal experiments [27]. In the present study, the expression levels of TLR4 were confirmed to be higher in adhesive tissues than in normal peritoneal tissues at 3, 5 and 7 days (Fig. 1A and C). In addition, IHF showed that the expression of TLR4 in peritoneal mesothelial cells was higher in adhesive tissues at 3, 5 and 7 days than in normal peritoneal tissues (Fig. 1B and D).

Fig. 1

TLR4 is highly expressed in adhesive tissues in a mouse model of postoperative intraperitoneal adhesion (four groups; n = 10 mice/group). (A) Typical IHC staining images of TLR4 on days 1, 3, 5 and 7 after surgery. Magnification, x200 (IHC); black lines indicate adhesive tissue and normal peritoneal tissue. (B) Representative IHF staining images of TLR4 (red fluorescence) on days 1, 3, 5 and 7 after surgery; CK19 (green fluorescence) was used as a marker of mesothelial cells. Magnification, x630 (IHF); yellow lines indicate adhesive tissue and normal peritoneal tissue. (C) IHC staining score of TLR4. ^*P < 0.05 compared with the relative control group; ^#P < 0.05 compared with the day 1 adhesive tissue group. (D) Expression of TLR4 in peritoneal mesothelial cells. ^*P < 0.05 compared with the relative control group; ^#P < 0.05 compared with the day 1 adhesive tissue group

The TLR4 inhibitor TAK-242 + HA can reduce abdominal adhesion formation in a mouse model

To further investigate the role of TLR4 in adhesion formation, mice that underwent postoperative abdominal adhesion surgery were treated with TAK-242 and HA. The results showed that the mice treated with TAK-242 + HA had lower adhesion scores than those in the control group (Fig. 2). To verify the effect of TAK-242, two other groups were treated with DMSO and TAK-242 + HA (Fig. S1), and it was revealed that TAK-242 + HA could effectively reduce the adhesion score.

Fig. 2

TAK-242 can attenuate postoperative abdominal adhesion formation in a mouse model (five groups; n = 8 mice/group). (A) Typical image of adhesive conditions in the different groups. The yellow arrow indicates adhesive tissue. (B) Nair’s score in the different groups. ^*P < 0.05, ^**P < 0.01 compared with the control group. (C) Non-adhesion rate in the different groups. ^*P < 0.05 compared with the control group

The TLR4 inhibitor TAK-242 + HA can reduce inflammatory reactions in adhesive tissues

Inflammation is the initial factor in the formation of adhesions. The present study examined inflammation-related indicators in different groups, and detected fewer inflammatory cells in TAK-242 + HA -treated adhesive tissues compared with those in the control group, and the inflammation score was lower than that in the control group (Fig. 3A and B). TGF-β1 and IL-6 are common inflammatory factors; the present study showed that TAK-242 and HA treatment could reduce the levels of TGF-β1 and IL-6 in adhesive tissues (Fig. 3C and D).

Fig. 3

TAK-242 can reduce inflammation in adhesive tissue (five groups; n = 8 mice/group). (A) Typical images of H&E staining in the different groups. The area between the black lines is adhesive tissue. Magnification, x200. (B) Inflammation score in the different groups. ^*P < 0.05, ^**P < 0.01 compared with the control group. (C) TGF-β1 levels in the different groups. ^*P < 0.05, ^**P < 0.01 compared with the control group. (D) IL-6 levels in the different groups. ^*P < 0.05, ^**P < 0.01 compared with the control group

The TLR4 inhibitor TAK-242 + HA can alleviate collagen deposition in adhesive tissues

Collagen deposition is the final process in the formation of adhesions. Sirius red staining and Masson staining showed that collagen thickness in TAK-242 + HA -treated adhesive tissues was lower than that in the control group (Fig. 4A-C). α-SMA is a marker of myofibroblasts, and the present study showed that TAK-242 and HA treatment inhibited α-SMA expression in adhesive tissues (Fig. 4A and C).

Fig. 4

TAK-242 can alleviate collagen deposition in adhesive tissues (five groups; n = 8 mice/group). (A) Typical images of Sirius red staining, Masson staining and IHC staining of α-SMA in different groups. Magnification, x200, the area between the black arrows indicates adhesive tissue. (B) Fiber thickness in different groups as assessed by Sirius red staining. ^*P < 0.05, ^**P < 0.01 compared with the control group. (C) Fiber thickness in different groups as assessed by Masson staining. ^*P < 0.05, ^**P < 0.01 compared with the control group. (D) IHC staining scores of α-SMA in the different groups. ^*P < 0.05, ^**P < 0.01 compared with the control group

The TLR4 inhibitor TAK-242 + HA can reduce ROS levels in adhesive tissues

Oxidative stress is an important factor that promotes the inflammatory response. The present study demonstrated that TAK-242 and HA could reduce ROS levels in adhesive tissues (Fig. 5A and B). Subsequently, the ROS-related factor MDA was examined in the different groups, and it was shown that MDA levels were lower in the adhesive tissues (Fig. 5C).

Fig. 5

TAK-242 treatment alleviates ROS levels in adhesive tissues (five groups; n = 8 mice/group). (A) Typical images showing ROS accumulation in the different groups on day 7 after surgery (red fluorescence). The nuclei were stained blue with DAPI. Magnification, x200; the area between the yellow areas indicates adhesive tissue. (B) ROS levels in the different groups. ^*P < 0.05 compared with the control group. (C) MDA levels in the different groups; ^*P < 0.05 compared with the control group

Discussion

The present study revealed that the TLR4 inhibitor TAK-242 in combination with HA alleviated abdominal adhesion formation in a mouse model, possibly by inhibiting inflammation and ROS levels, and reducing collagen deposition. Although the statistical significances in the HA group and the TAK-242 group (compared with the control group) were not achieved, a declining trend could be observed. This finding may improve the understanding of the formation of postoperative abdominal adhesions and provides a theoretical basis for using TAK-242 as an agent to prevent postoperative abdominal adhesion development.

In the past decade, immune checkpoint inhibitors (ICIs) have developed rapidly and have yielded promising results in patient care [31]. As a new type of immune checkpoint, targeted drugs for TLRs are undergoing preclinical and clinical research, and have great application prospects. TLR-4 signaling is one of the major pro-inflammatory pathways activated by exogenous pathogen-related or endogenous danger-related molecules, and therefore manipulation of TLR4 pathways is considered to have great therapeutic potential. Previously, Li et al. demonstrated that TLR4 expression and NF-κB activation were elevated in pancreatic tissue using a murine model of pancreatitis [32]. In patients with COVID-19, TLR4 activation and TLR4 signaling aberration have been shown to lead to myocarditis and multiple-organ injury [33]. Hua et al. revealed that TLR4-mediated signaling was activated after ischemia, and contributed to increased inflammatory responses and further brain injury [34,35,36]. In the formation of postoperative abdominal adhesions, inflammation is the initiating factor and persists until firm adhesions are formed [37]. The present study, consistent with the results of previous studies, verified that TLR4 was highly expressed in adhesive tissues, and demonstrated that the TLR4 antagonist TAK-242 in combination with HA could alleviate inflammation in adhesive tissues. Notably, TAK-242 was applied to damaged peritoneal wounds, which is an innovation of the present study. During the formation of adhesions, the exuded fibrous material is gradually degraded within 3–5 days after surgery by the fibrinolytic system and the extracellular matrix metalloenzyme system; otherwise, firm adhesions are formed by the secretion of collagen by fibroblasts [16]. Dysregulated collagen deposition can lead to complications, such as biomaterial fibrosis, cardiac fibrosis, desmoplasia, liver fibrosis, and pulmonary fibrosis, which can ultimately result in losses of organ function or failure of biomedical implants [38, 39]. Ekihiro et al. generated a mouse model of hepatic fibrosis and found that TLR4-mutant mice had a significant reduction in fibrosis, as demonstrated by Sirius red staining, hydroxyproline content and expression of α-SMA [40]. During peritoneal adhesion formation, fibroblasts in the microenvironment of adhesion tissue are activated and produce collagen and extracellular matrix deposits. If the tissue adhesion is severe or long-lasting, collagen deposition exceeds collagen degradation and finally leads to fibrosis [41, 42]. Our research also revealed that the TAK-242 had potential to alleviate collagen deposition and inhibit α-SMA expression in adhesive tissues. Which may provide new inspiration for preventing fibrotic disease. Besides, the TAK-242 was applied to damaged peritoneal wounds, which is another innovation of the present study.

Oxidative stress is caused by an imbalance between the production of free radicals and antioxidant enzymes [43]. Previous studies have demonstrated that TAK-242 can reduce ROS and protect against IRI in liver transplantation [26]. Correspondingly, it has also been reported that polystyrene microplastics (PS-MPs) treatment can upregulate the expression of TLR4 and NOX2, and the levels of ROS. Conversely, inhibition of the TLR4/NOX2 signaling pathway may effectively reduce ROS levels in cells [44]. Xia et al. demonstrated that TAK-242 could improve oxidative stress and mitochondrial dysfunction by inhibiting the activity of TLR4/NF-κB signaling pathway, thereby preventing sepsis-associated acute kidney injury in rats [45]. During the formation of postoperative abdominal adhesions, a large amount of ROS is generated due to the destruction of local tissues, which in turn aggravates the inflammatory response and leads to tissue damage. The present study showed that ROS levels were lower in the TAK-242-treated group than those in the control group, which may be another reason for TAK-242-mediated attenuation of adhesion formation.

Notably, the present study has some limitations. First, it is unclear whether TAK-242 directly inhibits the dimerization of TLR4 or the interaction of TLR4 with its adaptors [46]. Previous studies have suggested that Cox-2 and Ligustrazine nanoparticles (LN) could reduce postoperative peritoneal adhesion by targeting TLR4 through NF-κB pathway axis, suggesting that NF-κB is a promising target in the pathway of adhesion formation induced by TAK-242 and HA [47, 48]; therefore, further experiments are needed to explore the specific molecular mechanism. Second, Previous research indicates that the pro-inflammatory signaling events such as ERK/MAPK pathway, TLR cascades operating downstream of cluster 1 transcriptional modules have been shown to be active during the early fibrotic stages. While in the later stages, the cluster 2 transcription factors operating are enriched in physical interactions with chromatin remodelers, which have been shown to modulate global gene expression levels via histone modification-mediated chromatin remodeling [49, 50]. It is therefore unlikely that the TLR4 inhibitor, TAK-242, could be equally effective in the later stages of the phenomenon, where inflammatory signaling is less active than in the early and intermediate stages. Third, transgenic mice with TLR4 knockout were not used in the present study to confirm the role of TLR4. Forth, we have detected TNF-α, IL-6, and NF-κB at protein levels, which are closely related to inflammation and fibrosis in this experiment. Lamentably, we overlooked the expression of pro-inflammatory cytokine expression at mRNA levels due to the imperfections of our experimental designs.We also hope that in the future, we will have the opportunity to conduct further related research and investigations. Fifth, limited by the sample size and groups, the long-term effects and other administration methods of TAK-242 on post-operative adhesion have not been investigated. Further studies are needed to redeem these shortcomings. In addition, although the effect of TAK-242 in preventing peritoneal adhesion has been verified in animals, further prospective clinical trials are still needed to verify the effect on abdominal adhesion treatment.