*Contributed equally
Hepatic fibrosis is a global health problem, with increasing evidence demonstrating that oxidative stress serves a pivotal role in fibrogenesis. Riboflavin is a vital nutrient in the human and animal diet, which enhances the activity of antioxidant enzymes and ameliorates oxidative stress. The present study evaluated the effect of riboflavin on liver fibrosis and the mechanisms underlying this process. Rats were subcutaneously injected with carbon tetrachloride (CCl4) dissolved in sterile olive oil twice per week to induce hepatic fibrosis. The effect of riboflavin on CCl4-induced liver fibrosis was then assessed. Blood samples and liver tissues were collected and analyzed. The liver tissue morphological changes, immunohistochemical analysis, levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in the mitochondria, and the protein expression levels of α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK), AMP-activated protein kinase (AMPK), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and heme oxygenase 1 (HO-1) in the liver were also analyzed. The results demonstrated that riboflavin treatment significantly decreased the levels of alanine transaminase and aspartate transaminase in the serum, increased SOD activity and modulated the MDA level in the mitochondria. Furthermore, riboflavin significantly inhibited the CCl4-induced, upregulated protein expression levels of phosphorylated (p)-ERK, p-p38, p-JNK, TGF-β1 and α-SMA. Moreover, riboflavin significantly increased the expression of p-AMPK, PGC-1α and HO-1 in the liver tissue. These results suggested that riboflavin delays CCl4-induced hepatic fibrosis by enhancing the mitochondrial function via the AMPK/PGC-1α/HO-1 and mitogen-activated protein kinase signaling pathways.
Long-term liver injury caused by viral, alcoholic and drug is prevalent in the world, and almost 40% of patients further develop liver fibrosis (
The mitochondrion is a vital organelle in eukaryotic cells, supplying the energy for numerous biological functions via oxidative phosphorylation. Furthermore, mitochondria serve an essential role in the maintenance of various functions, including regulating the production of oxygen free radicals, calcium homeostasis and lipid metabolism (
Riboflavin, also called vitamin B2, is a heat-stable vitamin widely present in numerous foods, including milk, fish, dark-green leafy vegetables, fruits and rice (
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 (
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
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 (
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 (
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 (
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 (
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;
At the beginning of the experiment, there was no statistical difference in rat weight when comparing among the three groups (P>0.05;
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;
The level of blood glucose was not significantly different when compared among the 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;
Histological examination is one of the best methods for evaluating the severity of hepatic fibrosis (
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 (
The protein expression levels of p-AMPK
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 (
Previous research has demonstrated that CCl4 induces acute and chronic liver injury, including hepatic fibrosis, via numerous mechanisms, and thus, is considered a hepatotoxin (
Excessive production of ROS drives liver fibrosis via acceleration of the activation of HSCs (
The mitochondrion serves an essential role in the regulation of cellular metabolism through oxidative phosphorylation, and riboflavin participates in mitochondrial processes such as the metabolism of amino acids and fatty acids (
The MAPK signaling pathway regulates numerous cellular processes, including cell proliferation, differentiation and metabolism (
However, there are several limitations that need to be further explored to clarify the mechanism underlying the effect of riboflavin on CCl4-induced liver fibrosis. Firstly, the effect of inhibiting the AMPK/PGC-1α/HO-1 signaling pathway on liver fibrosis should be further evaluated. Secondly, the mechanisms underlying the effect of riboflavin on HSC activation and the related signaling should also be explored using an
In conclusion, the present study demonstrated that riboflavin attenuated CCl4-induced liver fibrosis via the AMPK/PGC-1α/HO-1 signaling pathway. Furthermore, riboflavin alleviated oxidative stress and decreased the expression of TGF-β1 and α-SMA in the liver via upregulation of the expression of AMPK, PGC-1α and HO-1, and downregulation of MAPK expression via the AMPK/PGC-1α/HO-1 signaling pathway. These findings suggest that riboflavin is a potential candidate for treating chronic liver injury.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
NT and WL performed the laboratory experiments. FH, WH and TTY performed the tissue analyses. GGW, FH and WL collected the data. GGW, NT and WL designed the experiments. GGW, NT, FH and WL analyzed the data. NT, WL and FH supervised the project. FH, GGW, WL and NT confirm the authenticity of all the raw data. GGW, WL and FH drafted the manuscript. All the authors read and approved the final manuscript.
The protocols for the experiments were approved by the Animal Experimental Ethics Committee of Wannan Medical College (approval no. LISC-2018-001).
Not applicable.
The authors declare that they have no competing interests.
Experimental design. Weight-matched rats were randomly allocated to three groups (n=10) and treated as indicated. CON, control; MOD, model; RIB, riboflavin; CCl4, carbon tetrachloride.
Histopathological changes in the liver of rats. Liver tissues were collected at the end of the experiment. (A) Changes in the liver tissues were assessed. Tissues were cut into thin sections and stained with (B) hematoxylin and eosin, and (C) Masson's Trichrome. Scale bar, 100 µm. CON, control; MOD, model; RIB, riboflavin.
Changes in body and liver weight and liver weight/body weight ratio. (A) Body weight and (B) liver weight of the rats. (C) Liver weight/body weight ratio. Values are presented as mean ± SD. *P<0.05, **P<0.01 vs. CON; $$P<0.01 vs. MOD (n=8). CON, control; MOD, model; RIB, riboflavin.
Parameters of liver function in serum. The serum (A) ALT and (B) AST levels were determined using an automated biochemical analyzer. Values are presented as mean ± SD. **P<0.01 vs. CON; $$P<0.01 vs. MOD (n=8). ALT, alanine transaminase; AST, aspartate aminotransferase; CON, control; MOD, model; RIB, riboflavin.
Changes of oxidation in the liver mitochondria. The (A) SOD activity and (B) MDA level in the liver mitochondria. Values are presented as mean ± SD. **P<0.01 vs. CON; $$P<0.01 vs. MOD (n=8). SOD, superoxide dismutase; MDA, malondialdehyde; CON, control; MOD, model; RIB, riboflavin.
Immunohistochemical analysis and expression of α-SMA and TGF-β1 proteins. The staining of (A) α-SMA and (B) TGF-β1 in liver tissues. Scale bar, 100 µm. (C) Western blotting of α-SMA and TGF-β1 in liver tissues. Relative protein expression levels of (D) α-SMA and (E) TGF-β1 in liver tissues. Values are presented as mean ± SD. **P<0.01 vs. CON; $$P<0.01 vs. MOD (n=6). α-SMA, α-smooth muscle actin; CON, control; MOD, model; RIB, riboflavin.
Effects of riboflavin on the expression of p-AMPK, PGC-1α and HO-1 proteins. (A) Western blotting of p-AMPK, PGC-1α and HO-1 protein in the liver. Relative expression of (B) HO-1, (C) PGC-1α and (D) p-AMPK in the liver. Values are presented as mean ± SD. **P<0.01 vs. CON; $$P<0.01 vs. MOD (n=6). p-, phosphorylated; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1α; HO-1, heme oxygenase 1; AMPK, AMP-activated protein kinase; CON, control; MOD, model; RIB, riboflavin.
Effects of riboflavin on the expression of MAPK signaling pathway-related proteins. (A) Western blotting of p-JNK, p-ERK and p-p38 in liver tissues. Relative protein expression levels of (B) p-JNK, (C) p-ERK and (D) p-p38 in the liver. Values are presented as mean ± SD. **P<0.01 vs. CON; $$P<0.01 vs. MOD (n=6). JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-related kinase; p-p38, phosphorylated; CON, control; MOD, model; RIB, riboflavin.
Effects of riboflavin on food consumption, blood glucose and epididymal adipose.
Parameter | CON | MOD | RIB |
---|---|---|---|
Food consumption, g/day | 23.64±1.85 | 18.69±1.49 |
19.94±1.72 |
Blood glucose, mM | 4.11±0.65 | 4.53±0.41 | 4.33±0.51 |
Epididymal adipose, g | 3.08±0.50 | 2.17±0.39 |
2.21±0.44 |
Epididymal adipose to BW, % | 0.69±0.08 | 0.60±0.09 |
0.59±0.10 |
Values are presented as mean ± SD.
aP<0.01 vs. CON;
bP<0.01 vs. MOD (n=8). CON, control; MOD, model; RIB, riboflavin; BW, body weight.