During the research period, all Ethical Considerations and Work Protocols on Laboratory Animals were in accordance with the recommendations established by the Laboratory Animal Ethics Committee and Supervision of Islamic Azad University, Tehran-North Branch, Tehran, Iran, as well as US NIH Guidelines (National Research Council of USA, 1996).The protocols were approved by the Ethics Committee of Islamic Azad University, Tehran-North Branch, Tehran, Iran. As well as, many efforts were made to minimize suffering, and all surgeries were performed under deep anesthesia. The study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).
Fifty-four adult male Wistar rats weighing approximately 230-250 g were purchased from the Pasteur Institute of Iran and transferred to the animal room of the Faculty of Basic Science, Islamic Azad University, Tehran-North Branch, Tehran, Iran with Ethics committee number IR.IAU.TNB.REC.1400.052. The animals were kept in a laboratory animal breeding center with fiberglass cages and under standard conditions with a temperature of 22 ± 2 ° C and a light cycle of 12 hours of light and 12 hours of darkness and relative humidity of 40-60%. For feeding the animals, special food for rats (plates) was prepared from Pars Animal Feed Company of Tehran and clean piped water was provided to the animals through drinking glasses without any restrictions.
Experimental design and BDL operation
After accommodation for one week, the rats were randomly classified into 9 groups of six animals each as follows: Group 1 (control): healthy intact animals that received daily distilled water as a drug solvent. Group 2 (Sham): the operated animals without BDL surgery which received distilled water daily as a drug solvent. Group 3 (BDL): Animals have undergone BDL surgery which received distilled water daily. Groups 4-6 (Experimental treatment): BDL-operated animals received daily treatment of brown algae extract at doses of 50, 100 or 200 mg/kg bw. Group 7-9 (Nizimuddinia control): intact animals that received daily treatment of brown algae extract at doses of 50, 100 or 200 mg / kg bw. The animals received brown algae extract dissolved in distilled water once a day by intragastric gavage. The volume of treatment was 0.5 ml and the duration of treatment was 45 consecutive days [27, 28]. Bile duct ligation (BDL) surgery was performed according to the standard method of Uchinami et al . Briefly, each animal was completely anesthetized by intraperitoneal (ip) injection of a mixture of ketamine (90 mg/kg bw) and xylazine (10 mg/kg bw). An abdominal incision was made from the midline. The common bile duct was identified and doubled ligated with a 4-0 nylon suture (AILEECo. Ltd., Busan, Korea) at two points (just below the junction of the hepatic duct and before the entrance to the pancreatic duct). The bile duct was then cut between these two points. At the end of the operation, 2 ml of sterile saline was injected into the peritoneum, followed by precise sutures of the peritoneum and muscles, as well as skin wounds. The animals were then allowed to recover on a heat pad . In sham rats, an abdominal incision was made without ligation of the common bile duct.
Preparation of Nizimuddinia zanardinii extract
The algae was prepared from Chabahar Port, Sistan and Baluchestan, Iran. The collected algae were stored in an ionolytic box containing ice and then transferred to the laboratory. The algae were thoroughly washed and immersed in distilled water (to remove solutes), and their water were changed for several hours. This was repeated up to three times, after which the algae were dried and completely pulverized by an electric mill. To prepare the ethanolic extract of the studied algae, 200 g of each algae powder sample was soaked in 80% ethanol solvent. Then, they were incubated for 24 hours at 100 rpm and room temperature. The extract extraction process was repeated two more times and then the extracts were filtered using Whatman No. 1 filter paper; Solubilization was performed under the hood and the extract was concentrated and kept at 4 ° C for later use [30, 31].
Sampling and biochemical evaluation
After the experimental period, the animals were fasted for 12 hours. Under anesthesia with i.p. injection of ketamine (90mg/kg bw) and xylazine (10mg/kg bw) the blood and livers were collected. After a 30-minute pause for blood clotting, the samples were centrifuged at 2500 rpm for 5 minutes to separate the serum from the blood and the serum levels of ALT, AST, ALP, cholesterol, triglyceride, total bilirubin, albumin and total protein were determined based on IFCC method (International Federation of Clinical Chemistry and Laboratory Medicine) and the utilized kits for evaluating the biochemical parameters by autoanalyzer device (BT1500, Italy) .
Measurement of hepatic superoxide dismutase (SOD) and catalase activities (CAT)
A part of the liver tissue of animals in all groups as fixed in 10% formalin buffer solution to prepare paraffin blocks from them after performing conventional tissue processing methods. Another part of the liver was used to prepare liver homogenate. The prepared homogeneous solutions were then poured into several follicles (equal volumes) and centrifuged by refrigerated centrifuge at 4 °C and 9000 rpm for 20 minutes. The milky supernatant was then collected to assay the activities of CAT and SOD activity was determined using the CAT and SOD assay kit (Pars Azmoon, Tehran, Iran) according to the manufacturers’ protocol that was assayed as described previously . Briefly, 0.2 ml of homogeneity was added to 1.2 ml of 50 mM phosphate buffer (pH = 7.5), the reaction starting with the addition of 1.0 ml of 30 mM H2O2 solution. Absorption reduction was measured at 240 nm at 30-second intervals for 30 minutes. A unit (U) of the activity of this enzyme is defined as the amount of enzyme that obtains the value K = 1, where K is the rate constant of the enzyme. Activity is expressed as a unit per mg of protein (U/mg protein) .
Trichrome staining is used to detect increased collagen deposition and to determine the severity of fibrosis in the liver tissues. 4-6 μm-thick sections cut from the liver of all groups were placed in xylene (2.5 minutes) and descending alcohol series (100, 90, 80, 80, 70, and 50 %) for deparaffinization. Then, the Sigma-Aldrich Masson’s Trichrome Stain Kit (HT15) was applied to the sections. They were washed with alcohol and xylene and covered with Entellan and light microscopic examinations were performed in a blinded fashion [34, 35]. The pathological findings were qualitative, and the liver damage in different groups was scored based on a modified method by Sant’Anna et al  using trichrome staining. In this method, 10 areas were randomly examined in each sample and the average injury was considered as a single score. The damage rating is as follows: necrosis: 0 = no necrosis, 1 = local damage less than 25%, 2 = local damage between 25-50%, 3 = extensive but local necrosis, 4 = extensive necrosis; infiltration of inflammatory cells: 0 = no inflammation, 1 = focal inflammation less than 25%, 2 = focal inflammation between 25-50%, 3 = extensive but localized inflammation, 4 = extensive inflammation; connective tissue: 0 = no connective tissue, 1 = collagen deposition without a wall mattress, 2 = incomplete wall mattress, 3 = full and thin wall, 4 = full and thick wall; bile duct hyperplasia: 0 = no hyperplasia, 1 = less than 25% per lobule, 2 = 25-50% per lobule, 3 = extensive but localized, 4 = extensive .
Immunohistochemistry staining was performed on liver tissue sections to determine alpha smooth muscle actin (α-SMA), the most commonly used protein that is increased in liver fibrosis, and transforming growth factor-β1 (TGFβ1), which is increased in liver injury are considered liver injuries. Liver sections were used and dehydrated before epitope recovery in novocaster solution in toluene (Leica Biosystems, Germany). After neutralizing the endogenous peroxidase with 3% H2O2 for 10 minutes, the sections were incubated with blocking solution at 4 ° C with anti-TGF-β1, α-SMA antibodies (1: 100, dilution). The detection was performed using a polymer detection system (Novolink max polymer detection system, Novocastra Leica Biosystems) and DAB (3,30-Diaminobenzidine, Novocastra Leica Biosystems) as the chromogenic substrate. Hematoxylin staining was used before dehydration and installation. Images were assessed by light microscopy (Olympus BX43, Hamburg, Germany) .
All data were statistically analyzed by SPSS ver20 using one-way analysis of variance (One-way-ANOVA) and Tukey post hoc test. The results are presented as Mean ± S.E.M. P<0.05 was considered statistically significant.