Subjects
For in vivo studies, we used 3-month-old Wistar rats with body weight of 250-300 g. Rats were obtained from the Department of Pharmacology and Therapy Faculty of Medicine, Public Health and Nursing Universitas Gadjah Mada, Indonesia. Our research protocol referred to the provisions of the principles of handling experimental animals and has obtained ethical permission about research using experimental animals from our institution's ethics commission. All animals in our study were maintain in international standard animal facility in the best possible conditions and got the best possible care from skilled and experienced animal caregiver. They were acclimatized for 7 days with controlled room temperature and received a regular 12/12 hr lighting cycle. Experimental animals were given standard feed and water ad libitum. For the in vitro studies, fibroblasts were primary isolated from the colons of healthy Wistar rats.
Treatment
Rats were divided into 3 groups of 6 rats each. All groups underwent intestinal anastomosis surgery. After the operation, each group received a different analgesic therapy. One group served as the control which only received aquadest therapy, while the other two groups received either metamizole therapy (60 mg/kg/day) or paracetamol (60 mg/kg/day) as previous study[9]. For in vitro studies, rat colon fibroblasts were cultured with the number of cells each of 1.75 x 105 and divided into 3 groups, which were the control, metamizole and paracetamol groups with 3 different doses each (250 µg/mL, 50 µg/mL, and 5 µg/mL).
Operating procedure
Anastomotic operations were conducted under sterile conditions. Rats were anesthetized using intramuscular anesthesia containing 0.5 ml ketamine (100 mg/ml), 0.125 ml xylazine (20 mg/ml), 0.075 ml acepromazine (10 mg/ml) and 3 ml sterile saline at 0.1 ml/100 g body weight . All rats received midline 2 cm laparotomy followed by 0.5 cm intestine resection. All resections were performed to intestinal section 5 cm distal from the caecum. The intestinal connection was done with end to end anastomosis with all 5-8 layers inverted, with interrupted sutures. The abdominal wall was closed by simple interrupted suture. After the operation, each rat received an analgesic according to the group: control, paracetamol, and metamizole. After 3 days post-operation, rats were euthanized using high dose of anesthesia containing ketamine, xylazine, and acepromazine at 3 times higher than normal dose (0.3 ml/100 g body weight). We proceeded to sacrifice the rats until no heart beat was detected for at least 5 minute. Furthermore, the intestinal segment with anastomosis was removed for further examination.
Intestinal anastomosis assessment
The intestinal anastomosis was assessed by scoring the integrity of muscle tissue, granulation tissue, and mucous anastomosis. The examination was conducted on histological preparations. The intestines with anastomosis were made into paraffin blocks then hematoxylin-eosin stained to determine sample histology in general. The integrity of the colon muscle tissue and the mucosal anastomosis were assessed and scored. Granulation tissue was assessed by the infiltration of inflammatory cells in the anastomosis area.
Fibroblast activities measurement
Activation of fibroblasts was assessed from proliferation, migration, and synthesis of collagen. Proliferation was assessed by comparing the level of IC50. Migration was assessed by calculating the difference between before and after injuring fibroblast cultures with the scratch assay method. Collagen synthesis was determined by calculating the absorbance of Sirius red staining on fibroblasts.
Fibroblast migration test with scratch wound assay
Wounding on cultured cells was done by scraping fibroblast cells in each well using 10-200 μL tip pipettes or blue micropipettes then incubating at 37°C, 10% CO2 for 1x24 hours. After incubation, the well was washed with PBS twice and 500 μL of Meyer hematoxylin were added in each well then incubated at room temperature for about 1 minute. Furthermore, each well was filled with 1 mL phosphate buffered saline (PBS), then microscopic images were converted to JPEG format, and empty space pixels and white pixels were calculated with ImageJ software.
Fibroblast cell proliferation test
Cells were incubated for 24 hours. Next, the appropriate treatment for each well was added: 0.9% saline or paracetamol (concentration 250 µg/mL, 50 µg/mL, or 5 µg/mL) or metamizole (concentration 250 µg/mL, 50 µg/mL, or 5 µg/mL). The control group received sterile aquadest treatment. The cells were incubated again for a specified time, which was 48 hours. The culture media in each well were transferred into microtubes. Pepsin was added to each well and incubated for 10 minutes, then the solution from each well was transferred to the appropriate microtube. For each cell suspension microtube, 5 µL cell suspension was taken and then mixed with 5 µL trypan blue. The number of cells in the mixture was calculated using a counting chamber under a microscope.
Each treatment with NSAIDs was done on a triplicate basis and repeated three times. After 48 hours of incubation, the numbers of live and dead fibroblasts in each treatment and control group were calculated. Afterward, the percentage of cell death in each well was calculated then converted to probit value. Next, a linear regression equation was made between the log concentration and the probit value so that the IC50 value was obtained as an antilog from the point where y=5. After that, the average IC50 of each NSAIDs type was calculated.
Collagen synthesis test
Cells were given each treatment for 24 hours, then the media was aspirated and washed with 200 µL PBS 3 times per well. After that, the well was fixed with a 100 µL Bovine solution for 1 hour. Then, the well was washed with distilled water until clean and dried overnight. Next, a solution of 100 µL of Sirius Red was given in each well and incubated for 1 hour. Next, the Sirius Red dye was removed and the well was washed with 100 µL 0.1 N HCl for 2-3 times. Then, the HCl was removed and washed until Sirius Red's solution was cleared. 200 µL of 0.5 N NaOH was added to the well and was left for 30 minutes. The absorbance reading was conducted at a wavelength of 570 nm with a plate reader.
Data Analysis
Data analysis was conducted using the SPSS Statistics 17.0 for Windows application. The data obtained were tested for normality in advance by the Kolmogorov Smirnov test to determine whether the data was normally distributed. If the data were normally distributed, the student's t-test was done, and if they were not normally distributed, the Mann-Whitney U-test was used.