Riboflavin was purchased from Nanjing Chemical Reagent Co., Ltd. The bicinchoninic acid (BCA) kit was purchased from Beyotime Institute of Biotechnology. Alanine transaminase (ALT) microplate assay kit (cat. no. C009-2), aspartate transaminase (AST) microplate assay kit (cat. no. C010-2), glucose assay kit (cat. no. F006-1-1), malondialdehyde (MDA) assay kit (TBA method; cat. no. A003-1) and superoxide dismutase (SOD) assay kit (Hydroxylamine method; cat. no. A001-1) were purchased from Nanjing Jiancheng Bioengineering Institute. Standard laboratory rodent food was purchased from Nanjing Qinglongshan Experimental Animal Feed Technology Co. Carbon tetrachloride (CCl₄) was purchased from Sinopharm Chemical Reagent Co., Ltd. Primary polyclonal antibodies, including AMP-activated protein kinase (AMPK; cat. no. ab32047), phosphorylated (p)-AMPK (cat. no. ab133448) and peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α; cat. no. ab106814) were purchased from Abcam, heme oxygenase (HO)-1 (cat. no. 82206), c-Jun N-terminal kinase (JNK; cat. no. 9252S), p-JNK (cat. no. 4668P), extracellular signal-regulated kinase (ERK)1/2 (cat. no. 4695S), p-ERK1/2 (cat. no. 4370P), p38 (cat. no. 8690S) and p-p38 (cat. no. 9211S) were purchased from Cell Signaling Technology Inc., and β-actin (cat. no. bs-0061R), α-smooth muscle actin (α-SMA; cat. no. bs70000) and transforming growth factor β1 (TGF-β1; cat. no. bs1361) were purchased from Bioworld. HRP-conjugated goat anti-rabbit IgG secondary antibodies (cat. no. BST11112B54) and rabbit anti-goat IgG secondary antibodies (cat. no. BA1060) were purchased from Wuhan Boster Biological Technology, Ltd.

All animal experiments were performed in compliance with the Guide for the Care and Use of Laboratory Animals (30) and the experimental protocol was approved by the Animal Experimental Ethics Committee of Wannan Medical College (Wuhu, China; approval no. LISC-2018-001). The rats received humane care and all efforts were made to alleviate suffering.

A total of 30 male Sprague-Dawley rats (weighing 240-260 g; 8-10 weeks) were purchased from the Changsha Tianqin Biotechnology Co., Ltd.. The rats were housed in a standard facility at 22±2˚C with 50±5% humidity and a 12/12 h light/dark cycle. The animals had access to water and food ad libitum. After a week of acclimation, rats were randomly divided into groups (n=10) as follows: i) Control (CON) group; ii) model (MOD) group; and iii) riboflavin (RIB) group.

CCl₄ is catabolized into the trichloromethyl radical by cytochrome P450 2E1 (CYP2E1) in the liver. Trichloromethyl radicals and oxygen are further converted into the trichloromethyl peroxyl radical. These radicals cause liver injury, and chronic liver injury further progresses into hepatic fibrosis and cirrhosis of the liver (31). In addition to its anesthetic effect, phenobarbital enhances the activity of CYP2E1, promoting conversion of CCl₄ into the trichloromethyl radical and the subsequent development of hepatic fibrosis (32). Phenobarbital has been reported to upregulate CYP2E1 when administered via drinking water (33,34).

In the present study, liver fibrosis was induced according to the aforementioned methods with minor revisions. Briefly, the rats in the RIB and MOD groups received a subcutaneous injection of CCl₄ dissolved in sterile olive oil (0.4 ml/kg; v/v, 1/1) twice per week and phenobarbital (0.35 g/l) dissolved in drinking water for 12 weeks (Fig. 1). Oxidative stress was induced using riboflavin as previously described (24). Briefly, the RIB group received an oral riboflavin dose (20 mg/kg/day). The animals in the CON group received a subcutaneous injection of olive oil (0.2 ml) twice per week (Fig. 1). To observe effect of riboflavin on the state of rats treated with CCl4, general characteristics of the rats, including mental state, activity, and eating and drinking status, were observed every day.

The amount of food consumed every day was recorded. The rats were weighed once every 2 weeks over the experimental period. The rats were anesthetized after 12 weeks using an intraperitoneal injection of sodium pentobarbital (40 mg/kg). Blood (3 ml) was then collected from the carotid artery. To minimize pain, the rats were sacrificed via intraperitoneal injection with sodium pentobarbital (100 mg/kg) and bleeding. Rats were confirmed dead when there was no autonomous respiration and no reflex activity, and no heart activity. The liver, spleen and pancreas were immediately removed and weighed. A portion of the left lobe of the liver was removed and fixed in 10% neutral formalin for 2 days at room temperature. The remaining portion of the liver was stored at -80˚C until use. The epididymal adipose tissue was separated and weighed to assess the amount of visceral fat.

Blood samples were centrifuged at 10,000 x g at 4˚C for 10 min to extract the serum. The levels of ALT, AST and glucose levels in the serum were quantified using a Hitachi 7600-120 automated biochemical analyzer (Hitachi High-Technologies Corporation). Serum was mixed with 2,4-dinitrophenylhydrazine and incubated for 30 min at 37˚C, then 0.4 mol/l sodium hydroxide was added into mixture. Absorbance at 505 nm was used to measure AST and at 510 nm for ALT.

MDA (a product of lipid peroxidation) and SOD (an antioxidant enzyme) levels in the mitochondria were used to assess changes in the level of oxidative stress.

Mitochondria in the liver were separated as previously described (35). Briefly, liver tissues were immersed in a precooled buffer (10 mM Tris, 210 mM mannitol, 70 mM sucrose, 1 mM EDTA and 0.5 mM EGTA; pH 7.4) and immediately cut into 1-mm portions. Portions were homogenized and centrifuged at 1,000 x g at 4˚C for 10 min. The supernatant was collected and centrifuged at 10,000 x g for 15 min at 4˚C. The precipitate was resuspended in the buffer and the precipitate suspension was centrifuged at 10,000 x g and 4˚C for 15 min.

The precipitate was resuspended in normal saline to determine the levels of SOD and MDA in liver mitochondria using commercially available kits according to the manufacturer's protocol.

The liver was fixed in 4% neutral paraformaldehyde for 2 days at room temperature, embedded in paraffin after dehydration in an ascending ethanol series in turn (from 75 to 100% ethanol) and serially cut into 5-µm thick sections to observe morphological features and fibrosis. For histological examination, the sections were stained with hematoxylin (15 min) and eosin (3 min) (H&E) at 25˚C. For assessment of the presence of collagen in the livers, the sections were stained with Masson's Trichrome. Staining in Weigert hematoxylin for 8 min, ponceau for 10 min and aniline blue for 2 min at 25˚C.

Immunohistochemical staining of TGF-β1 and α-SMA was performed to examine the activated hepatic stellate cells (HSCs). The liver was embedded in paraffin and cut into 5-µm thick sections. After deparaffinization with xylene for 10 min and hydration in a descending alcohol series at room temperature, the sections were incubated with boiled 10 mM sodium citrate buffer for 5 min for epitope retrieval. The sections were treated with 3% hydrogen peroxide to inhibit endogenous peroxidase activity for 15 min at room temperature. The sections were washed with phosphate buffered saline (PBS) for 2 min three times at room temperature and the sections were treated with PBS containing 2% bovine serum albumin (BOMEI Biotechnology CO., LTD. Hefei, China) to block non-specific sites for 1 h at 37˚C. Sections were incubated with anti-α-SMA (1:100) and anti-TGF-β1 antibodies (1:100) overnight at 4˚C. The sections were then washed with PBS and incubated with HRP-conjugated goat anti-rabbit IgG secondary antibodies (1:100) for 1 h at 25˚C. Antigen staining was visualized using the Enhanced HRP-DAB Chromogenic kit (BaSo Biotechnology Co., Ltd.) and observed under a Moticam S6 microscope (Motic China Group Co., Ltd.).

Western blotting was performed as described previously (36). Briefly, liver tissues were lysed in ice-cold lysis buffer (10 mM HEPES, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 20 mM sodium fluoride, 10 mM EDTA, 2 mM PMSF, 1% Triton X-100) and centrifuged at 13,000 x g at 4˚C for 15 min. After quantification using the BCA method, the proteins (30 µg) in lysates were separated using a 10% SDS-PAGE gel and transferred onto nitrocellulose membranes. The membranes were blocked in 2% bovine serum albumin (dissolved in PBS; Hefei Bomei Biotechnology Co., Ltd.) for 1h at room temperature. Then, the membranes were incubated with the following specific antibodies: β-actin, HO-1, AMPK, p-AMPK, PGC-1α, ERK, p-ERK, JNK, p-JNK, p38, p-p38, TGF-β1 (all 1:1,000) and α-SMA (1:2,000) overnight at 4˚C. The membranes were washed three times with PBS and incubated with HRP-conjugated goat anti-rabbit IgG secondary antibodies (1:10,000) for 90 min at room temperature, and the membrane for PGC-1α, with rabbit anti-goat IgG secondary antibodies. Protein bands were visualized using the ECL chemiluminescence substrate kit (Beijing labgic Biotechnology CO., LTD. Beijing, China) according to the manufacturer's protocol and analyzed using Image J software (version 1.8; National Institutes of Health). β-actin was used as the loading control.

Data were analyzed using SPSS version 20.0 (IBM Corp.). Results are presented as the mean ± standard deviation. Differences between groups were analyzed using one-way ANOVA and Dunnett's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

After 4 weeks of treatment with CCl₄, the rats in the MOD group were depressed and dull. Administration of riboflavin markedly improved these characteristics. The rats in the MOD group were also less active and had a poor appetite. Food consumption was significantly higher for rats in the CON group than in the MOD group (P<0.01; Table I, the food consumption in the MOD group was significantly decreased compared with the RIB group (P<0.01). Granular protrusions were observed on the liver surface of rats in the MOD group (Fig. 2A) and fewer granular protrusions in the RIB group. Riboflavin supplementation improved the appetite of rats in the RIB group.

At the beginning of the experiment, there was no statistical difference in rat weight when comparing among the three groups (P>0.05; Fig. 3A). However, after 2 weeks of CCl₄ treatment, bodyweight in the CON group was significantly higher compared with that of rats in the MOD group (P<0.05). However, there was no significant difference in the body weight of rats in the MOD group compared with that in the RIB group (P>0.05). Further analysis demonstrated that there was no significant difference with regard to liver weight for rats in the three groups (P>0.05; Fig. 3B). However, the liver weight/body weight (LW/BW) ratio was significantly higher for rats in the MOD group compared with that in the CON group (P<0.05; Fig. 3C). Furthermore, riboflavin treatment significantly decreased the LW/BW ratio of the RIB group when compared with the MOD group (P<0.05). The results suggested that riboflavin attenuated liver fibrosis induced by CCl₄.

The liver function markers ALT and AST in the serum were quantified to assess the effects of riboflavin on liver function. The results demonstrated that the activities of ALT and AST were significantly higher in the MOD group compared with that in the CON group (P<0.05; Fig. 4) and that ALT and AST activities were significantly lower in the RIB group compared with the MOD group (P<0.05). These results demonstrated that oral administration of riboflavin significantly improved function impaired by CCl₄.

The level of blood glucose was not significantly different when compared among the groups (P>0.05; Table I). The level of epididymal fat, a marker of the visceral fat, was significantly higher in the CON group compared with that in the MOD group (P<0.05). Furthermore, the epididymal fat to BW ratio was significantly higher in the CON group compared with that in the MOD group (P<0.05); however, the ratio was not significantly different when compared between the MOD and RIB groups (P>0.05).

SOD exerts its antioxidant activity by scavenging superoxide. MDA, a lipid peroxidation product, reflects the oxidant-induced lipid peroxidation level. Therefore, SOD and MDA levels were quantified to evaluate oxidative stress. SOD activity in the liver was significantly lower in the MOD group compared with that in the CON group (P<0.01; Fig. 5A). Furthermore, the MDA level was significantly higher in the MOD group compared with that in the CON group (P<0.05; Fig. 5B). Treatment with riboflavin significantly decreased the MDA level (P<0.01) and significantly increased SOD activity in the liver (P<0.01) in the RIB group when compared with the MOD group. The results indicated that riboflavin ameliorated CCl₄-induced mitochondrial dysfunction in rats.

Histological examination is one of the best methods for evaluating the severity of hepatic fibrosis (37). For rats in the MOD group, the liver adhered to the surrounding tissues, and there is no obvious adhesion between the liver and surrounding tissues in the CON and RIB groups. The liver surface was significantly rougher and more nodular compared with the CON group. However, riboflavin treatment markedly reduced the number of diffuse nodules on the liver surface. H&E staining demonstrated a lobular architecture, clear sinusoids and hepatocytes radiating around the central vein for rats in the CON group (Fig. 2B). However, rats in the MOD group displayed a poor liver structure, infiltration of inflammatory cells, and swollen and deformed hepatocytes. However, riboflavin treatment reduced the infiltration of inflammatory cells and necrosis in the liver for rats in the RIB group compared with the MOD group. Furthermore, Masson's Trichrome staining demonstrated that the deposition of collagen fibers was higher in the MOD group compared with that in the CON group. However, riboflavin treatment markedly decreased the deposition of collagen fibers in the liver of rats in the RIB group compared with those in the MOD group (Fig. 2C). Therefore, riboflavin treatment attenuated CCl₄-induced liver injury in rats.

TGF-β1 is a key profibrogenic cytokine in hepatic fibrosis. HSCs activated via TGF-β1 serve a vital role in liver fibrogenesis by promotion of the production of α-SMA. Therefore, α-SMA is a marker of activated HSCs. Immunohistochemical staining demonstrated that CCl₄ treatment markedly increased the expression of TGF-β1 and α-SMA in the MOD group compared with that in the CON group (Fig. 6A and B). The administration of riboflavin markedly decreased the expression of TGF-β1 and α-SMA in the RIB group compared with that in the MOD group. The protein expression levels of TGF-β1 and α-SMA in the liver were assessed using western blotting (Fig. 6C). The results demonstrated that compared with the CON group, CCl₄ treatment significantly increased the protein expression levels of TGF-β1 and α-SMA in the MOD group (P<0.01; Fig. 6D and E). However, riboflavin treatment significantly decreased the protein expression levels of TGF-β1 and α-SMA compared with the MOD group (P<0.01). The results showed that riboflavin treatment attenuated CCl₄-induced liver fibrosis via decreasing the expression of fibrogenic cytokines.

The protein expression levels of p-AMPK, PGC-1α and HO-1 in the liver tissue were assessed (Fig. 7A) to demonstrate the antioxidant effect of riboflavin. CCl₄ treatment significantly decreased the expression of HO-1, PGC-1α and p-AMPK in the MOD group compared with that in the CON group (all P<0.01; Fig. 7B-D). However, riboflavin treatment significantly increased the protein expression levels of HO-1, PGC-1α and p-AMPK when compared with the MOD group (all P<0.01). The results indicated that riboflavin relieved liver fibrosis via the improvement of AMPK/PGC-1α/HO-1 signaling on mitochondrial function and oxidative stress.

The MAPK signaling pathway participates in the modulation of collagen expression and accelerates fibrogenesis. The protein expression levels of proteins related to the MAPK signaling pathway were assessed using western blotting to demonstrate the mechanism of riboflavin action on liver fibrosis (Fig. 8A). CCl₄ treatment of the MOD group significantly increased the protein expression levels of p-ERK, p-JNK and p-p38 compared with the CON group (all P<0.01; Fig. 8B-D). However, riboflavin treatment significantly decreased protein expression levels of p-ERK, p-JNK and p-p38 compared with the CON group (all P<0.01). The results suggested that riboflavin improved the MAPK signaling, which attenuated liver fibrosis.