Background: Didymin has been reported to have anti-cancer potential. However, the effect of didymin on liver fibrosis remains illdefined. Methods: Hepatic fibrosis was induced by CCl4 in rats. The effects of didymin on liver pathology and collagen accumulation were observed by hematoxylin-eosin and Masson's trichrome staining, respectively. Serum transaminases activities and collagen-related indicators levels were determined by commercially available kits. Moreover, the effects of didymin on hepatic stellate cell apoptosis and cell cycle were analyzed by flow cytometry. Mitochondrial membrane potential was detected by using rhodamine-123 dye. The expression of Raf kinase inhibitor protein (RKIP) and the phosphorylation of the ERK/MAPK and PI3K/Akt pathways were assessed by Western blot. Results: Didymin significantly ameliorated chronic liver injury and collagen deposition. It strongly inhibited hepatic stellate cells proliferation, induced apoptosis and caused cell cycle arrest in G2/M phase. Moreover, didymin notably attenuated mitochondrial membrane potential, accompanied by release of cytochrome C. Didymin significantly inhibited the ERK/MAPK and PI3K/Akt pathways. The effects of didymin on the collagen accumulation in rats and on the biological behaviors of hepatic stellate cells were largely abolished by the specific RKIP inhibitor locostatin. Conclusion: Didymin alleviates hepatic fibrosis by inhibiting ERK/MAPK and PI3K/Akt pathways via regulation of RKIP expression.

Liver fibrosis is reversible wound-healing response against a variety of acute and chronic stimulation in the liver, including ethanol, viral infection, cholestasis and metabolic disease [1,2]. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension [3]. It has been reported that the proliferation and activation of hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis [4], and targeting the proliferation of HSCs can be considered as novel therapeutic strategy for treating liver fibrosis [5].

Natural products are rich source of compounds with enormous structural diversity and have been extensively studied in the field of drug discovery. Didymin is a flavone that is common in citrus fruits like oranges, lemons, mandarin and bergamot. It can cause cell death in non-small cell lung cancer in a p53 independent manner [6]. Moreover, it can inhibit neuroblastomas proliferation and induces apoptosis by inhibiting N-Myc and up-regulating Raf kinase inhibitor protein (RKIP) level making it a potential new approach for neuroblastoma therapy [7]. Although the anti-cancer potential of didymin is well known, its precise mechanisms has not been investigated. Especially, the effect of didymin on liver fibrosis remains unclear.

To understand the anti-fibrosis effect of didymin, the present study was designed to investigate the effects of didymin on liver injury and collage deposition in vivo and in vitro. Additionally, to investigate whether didymin alleviated hepatic fibrosis through up-regulating Raf kinase inhibitor protein (RKIP), locostatin, a specific inhibitor of RKIP [8], was used to interfere with RKIP expression, and the antagonism between both the drugs was observed.

Materials

Mentha spicata L. was purchased from Nanning Qianjinzi Chinese Pharmaceutical Co. Ltd (Nanning, China). Locostatin was obtained from Merck (Darmstadt, Germany). Fetal bovine serum (FBS) was purchased from Life Sciences (Grand Island, NY). MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and propidium iodide (PI) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A).

Preparation of didymin

M. spicata L. herb (30 kg) was extracted with 95% ethanol under reflux three times for 1.5 h each time; the combined solvent was evaporated in vacuo to remove the ethanol. The extracts were suspended in methanol and fractionated with petroleum ether. The methanol fraction was subjected to silica gel chromatographic column (200-300 mesh, Yantai, PR China; 10×300 cm) and eluted with trichloromethane /methanol (100:0→50:50, each 500 mL) to produce eight fractions (Fr. 1- Fr.8). Fr. 7 was applied to a silica gel column (trichloromethane/methanol, 95:5→50:50) to generate eight fractions (Fr.7.1- Fr. 7.8). Fr. 7.4 was successively separated by silica gel column chromatography (trichloromethane/methanol, 80:20) to generate a white compound (128.6 mg). Its spectroscopic data such as ESI-MS, 1H-NMR and 13C-NMR were consistent with the data of didymin that was reported in the previous study [9], which indicates that the isolated compound is didymin.

Hepatic fibrosis model in rats and drug administration

Male SD rats, weighting 160 -180 g, were obtained from Guangxi Medical University Experimental Animal Center (Guangxi, China) and the research was conducted according to protocols approved by Guangxi Medical University Institutional Ethical Committee. Following one week acclimation period, rats were randomly divided into five groups (12 rats/group) including normal control group, didymin control group, CCl4 model group, CCl4+ didymin (CD) group and CCl4 + didymin + locostatin (CDL) group. The rats in the last three groups received 2 ml/kg CCl4 (mixed 1:1 in peanut oil) intragastrically twice a week for 12 weeks, while the animals in the normal and didymin control groups received equivalent peanut oil.

Additionally, during the period from weeks 8 to 12, the rats in the didymin control group and CD group were intraperitoneally administrated with 0.5 mg/kg didymin, and the rats in the CDL group received 0.5 mg/kg didymin plus 0.5 mg/kg locostatin once a day; while the animals in the normal control group and the CCl4 model group received equivalent normal saline. At the end of treatment, all animals were sacrificed and blood and liver samples were obtained for further examination.

Histological examination of liver tissues

Liver tissues were fixed in 10% formalin, embedded in paraffin and sectioned at 5-µm thickness. The liver pathology was observed by hematoxylin-eosin (H&E) staining. Additionally, the collagen deposition was observed by Masson's trichrome staining.

Serological tests and determination of collagen-related indicators

Serum alanine transaminase (ALT) and aspartate transaminase (AST) activities were measured using commercial kits (Nanjing Jiancheng Institute of Biotechnology, Nanjing, China), and TNF-α in plasma was detected using an enzyme-linked immunosorbent assay kit (Beijing Yonghui Biological Technology, Beijing, China). In addition, hydroxyproline (Hyp), type III precollagen (PCIII), laminin (LN) and hyaluronic acid (HA) were determined by commercially available kits (Nanjing Jiacheng Bioengineering Institute, Nanjing, China).

Cell line and cell proliferation assay

HSC-T6 cells were seeded in 96-well plates at a density of 1×105 cells/well. After 24 h incubation, cells were treated with 100, 50, 25, 12.5, 6.25 and 3.125 µM of didymin for 24, 48 and 72 h. Cell proliferation was then measured using MTT assay as previously described [9].

Cell apoptosis and cell cycle analysis

HSC-T6 cells were seeded into a 6-well plate at a density of 3× 105 cells/well and allowed to adhere overnight. Then cells were divided into four groups including normal control group (cells were treated with equal amount of culture medium), PDGF-BB group (cells were treated with 10 nM PDGF-BB [10]), didymin + PDGF-BB (DP) group (cells were treated with 25 µM didymin [6,7] + 10 nM PDGF-BB), and didymin + locostatin + PDGF-BB (DLP) group (cells were treated with 25 µM didymin + 25 µM locostatin [11] + 10 nM PDGF-BB ). HSC-T6 cells were treated with drugs for 24 h, and cell apoptosis and cell cycle were analyzed according to our previous study [9].

Mitochondrial membrane potential (MMP) analysis and detection of caspases activities

HSC-T6 cells were seeded in a 96 well plate at a density of 1 × 105 cells/well and administrated with drugs as described above for 24 h. Cells were added 5 mM rhodamine-123 dye (Rh-123, Sigma-Aldrich) for 30 min at room temperature in dark. MMP was then detected according to our previous study [9]. In addition, caspase-3 and -9 activities were detected using commercially available kits (BioVision Research Products, CA, USA) [9].

Western blot analysis

Whole cell proteins were prepared using RIPA buffer (Thermo Fischer Scientific, Inc., Waltham, MA) with 1% Halt protease inhibitor cocktail and 1% Halt phosphatase inhibitor cocktails (Thermo Fischer Scientific, Inc., Waltham, MA). Total hepatic proteins were extracted from liver tissues using radioimmunoprecipitation buffer containing a protease inhibitor cocktail (Sigma-Aldrich). The protein was separated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels, then transferred onto nitrocellulose membranes. The expression of proteins were detected using the following antibodies: RKIP, ERK, p-ERK, MEK, p-MEK, p38, p-p38, JNK, p-JNK, Bcl-2, Bax, cyclin D1, cyclin B1, CDK4, PI3K, Akt and p-Akt (Santa Cruz Biotechnology), respectively. All the primary antibodies were incubated overnight at 4 °C. The bands were scanned and quantified by NIH IMAGEJ 1.38 Software (US National Institutes of Health, Bethesda). The detected proteins were normalized to GAPDH (Santa Cruz Biotechnology) or the respective total protein, as appropriate.

Statistical analysis

Statistical analysis was performed using SPSS 11.5 for Windows. Differences between the groups were assessed using a one-way analysis of variance (ANOVA) with a Tukey's test for post hoc multiple comparisons. The data are presented as the means ± SD. A p-value < 0.05 was considered statistically significant.

Didymin alleviated liver injury and collagen deposition in rats

The effect of didymin on hepatic histological damage was assessed. The results showed that the livers in the normal and didymin control groups showed normal structure, and the hepatic cells were orderly arranged (Fig. 1 A1 and A2). In contrary, liver cells in the model group were edematous with fatty degeneration, and lobular architecture was destroyed or had disappeared (Fig. 1 A3). Didymin treatment significantly ameliorated histological damage, as evidenced by decrease in steatosis and hepatic lesions in the CCl4 + didymin group (CD group) (Fig. 1 A4). In addition, Masson's trichrome staining showed that the liver tissues in the normal and didymin control groups showed traces of collagen only in the walls of major blood vessels (Fig. 1 B1 and B2). However, CCl4 administration led to extensive accumulation of collagen in the liver tissues (Fig. 1 B3). Didymin treatment significantly decreased collagen deposition (Fig. 1 B4). Interestingly, the protective effect of didymin on liver injury (Fig. 1 A5) and collagen accumulation (Fig. 1 B5) were greatly abolished by the specific RKIP inhibitor locostatin.

Fig. 1

Didymin alleviated liver damage and collagen accumulation. (A) Liver injury was observed by Hematoxylin and eosin (H&E) staining; (B) Collagen distribution and deposition were observed by Masson's trichrome staining (200 ×). The arrows indicate hepatocellular necrosis in the H&E staining and collagen fibers in the Masson's trichrome staining, respectively. A1 and B1: normal control group; A2 and B2: didymin control group; A3 and B3: CCl4 model group; A4 and B4: CCl4 + didymin (CD) group; A5 and B5: CCl4 + didymin + locostatin (CDL) group. In addition, serum ALT and AST (C), plasma TNF-α (D), and the collagen-related indicators (E) were detected using commercial kits. Data were expressed as the means ± SD; aP<0.05 vs. the CCl4 model group; bP<0.05 vs. the CDL group.

Fig. 1

Didymin alleviated liver damage and collagen accumulation. (A) Liver injury was observed by Hematoxylin and eosin (H&E) staining; (B) Collagen distribution and deposition were observed by Masson's trichrome staining (200 ×). The arrows indicate hepatocellular necrosis in the H&E staining and collagen fibers in the Masson's trichrome staining, respectively. A1 and B1: normal control group; A2 and B2: didymin control group; A3 and B3: CCl4 model group; A4 and B4: CCl4 + didymin (CD) group; A5 and B5: CCl4 + didymin + locostatin (CDL) group. In addition, serum ALT and AST (C), plasma TNF-α (D), and the collagen-related indicators (E) were detected using commercial kits. Data were expressed as the means ± SD; aP<0.05 vs. the CCl4 model group; bP<0.05 vs. the CDL group.

Close modal

Didymin decreased the levels of ALT, AST and TNF-α

As shown in Fig. 1C, serum AST and ALT activities were significantly increased in the CCl4 model group compared to the normal control group. Treatment with didymin markedly decreased both enzymes activities, but its effect was partially abolished by locostatin. Similarly, didymin significantly decreased the content of TNF-α in the liver tissues, and the effect of didymin on TNF-α was reduced by locostatin (Fig. 1D).

Didymin decreased the levels of collagen-related indicators in liver tissues

The key collagen-related indicators including hepatic Hyp, PCIII, LN and HA were determined in this study. As shown in Fig. 1E, the contents of these indicators in the CCl4 model group were significantly higher than those of the normal control group. These up-regulations of Hyp, PCIII, LN and HA were markedly inhibited by didymin treatment. It was not surprise that the effect of didymin on the collagen-related indicators was largely abolished by locostatin.

Didymin inhibited cell proliferation

The effect of didymin on hepatic stellate cell proliferation was determined by MTT assay. As shown in Fig. 2A, didymin inhibited HSC-T6 cell proliferation in a distinct dose- and time-dependent manner. The 50% inhibitory concentrations (IC50) were 45.9 ± 2.7, 26.2 ± 1.6 and 16.6 ±1.4 µM after incubation for 24, 48 and 72 h, respectively.

Fig. 2

Effects of didymin on HSC-T6 cell proliferation, apoptosis and cell cycle. (A) Cell proliferation was detected by MTT assay. (B) HSC-T6 cell apoptosis and (C) cell cycle were analyzed by Flow cytometric assay. (D) Apoptosis-related proteins such as Bcl-2 and Bax, and (E) cycle-related proteins including CDK4, cyclin D1 and cyclin B1 were detected by Western blot. The bands 1 to 4 represented the normal control group, PDGF-BB group, DP group and DLP group. *P<0.05 vs. normal control group, #P<0.05 vs. PDGF-BB group and P<0.05 vs. DLP group.

Fig. 2

Effects of didymin on HSC-T6 cell proliferation, apoptosis and cell cycle. (A) Cell proliferation was detected by MTT assay. (B) HSC-T6 cell apoptosis and (C) cell cycle were analyzed by Flow cytometric assay. (D) Apoptosis-related proteins such as Bcl-2 and Bax, and (E) cycle-related proteins including CDK4, cyclin D1 and cyclin B1 were detected by Western blot. The bands 1 to 4 represented the normal control group, PDGF-BB group, DP group and DLP group. *P<0.05 vs. normal control group, #P<0.05 vs. PDGF-BB group and P<0.05 vs. DLP group.

Close modal

Didymin induced apoptosis and disturbed cell cycle distribution

To understand the underlying basis for the cytotoxic effect of didymin on HSC-T6 cells, cells apoptosis was analyzed by flow cytometry. The result showed that didymin significantly promoted HSC-T6 cell apoptosis compared to the PDGF-BB group, but this effect was largely abolished by locostatin (Fig. 2B). We next analyzed the effect of didymin on cell cycle progression by FACS analysis. The result showed that treatment with didymin led to a significant accumulation of cells in the G2/M phase, suggesting that didymin exerts its anti-proliferative effects by disturbing cell cycle distribution (Fig. 2C).

The regulation of didymin on apoptosis-related and cycle-related proteins

Bcl-2 family proteins serve as critical regulators of pathways involved in apoptosis, acting to either inhibit or promote cell death [12]. As shown in Fig. 2D, didymin treatment significantly decreased the anti-apoptotic protein Bcl-2 expression, while increased the pro-apoptotic protein Bax expression. However, the effects of didymin on the apoptosis-related proteins were largely abolished by locostatin. In addition, cell cycle is controlled by cyclin-dependent kinases (CDKs), cyclin kinase inhibitors and cyclins [13]. Our result showed that didymin treatment significantly reduced the expressions of the cell cycle-related proteins including cyclin B1, cyclin D1 and CDK4 (Fig. 2E).

Didymin induced the loss of MMP and the release of mitochondrial cytochrome c

The change of MMP was evaluated by flow cytometry using rhodamin-123 (Rh-123). As shown in Fig. 3A, the intensity of fluorescence in the DP group was significantly decreased compared to the PDGF-BB group, indicating that didymin treatment induced apoptosis of HSC-T6 cells by disruption of MMP. Additionally, we further analyzed cytochrome c (Cyt c) level by Western blot assay. As shown in Fig. 3B, didymin treatment decreased Cyt c level in the mitochondria with a corresponding increase in cytosolic level, indicating that Cyt c was released from the mitochondria into the cytosol. These findings suggest that mitochondrial dysfunction is likely involved in didymin-induced apoptosis. Interestingly, locostatin did not abolish the effect of didymin on MMP or Cyt c. These results suggest that the underlying mechanism of didymin on apoptosis is partially RKIP-independent.

Fig. 3

Didymin decreased MMP, promoted mitochondrial cytochrome c release, and increased caspase-3 and -9 activities in HSC-T6 cells. (A) The mitochondrial membrane potential (MMP) was monitored by staining with Rh-123 dye. Results were expressed as mean Rh-123 fluorescence. (B) Cytochrome c (Cyt c) expression was determined by western blot. The bands 1 to 4 represented the normal control group, PDGF-BB group, DP group and DLP group. (C) Caspase-3 and -9 activities were detected using commercially available kits. *P<0.05 vs. normal control group and #P<0.05 vs. PDGF-BB group.

Fig. 3

Didymin decreased MMP, promoted mitochondrial cytochrome c release, and increased caspase-3 and -9 activities in HSC-T6 cells. (A) The mitochondrial membrane potential (MMP) was monitored by staining with Rh-123 dye. Results were expressed as mean Rh-123 fluorescence. (B) Cytochrome c (Cyt c) expression was determined by western blot. The bands 1 to 4 represented the normal control group, PDGF-BB group, DP group and DLP group. (C) Caspase-3 and -9 activities were detected using commercially available kits. *P<0.05 vs. normal control group and #P<0.05 vs. PDGF-BB group.

Close modal

Didymin enhanced the activities of caspase-3 and -9

To fully understand the underlying mechanism of didymin on cell apoptosis, we also examined the protease activities of caspase-3 and -9. As shown in Fig. 3C, compared to the normal control group, PDGF-BB decreased both the protease activities. In contrast, didymin treatment significantly increased the activities of caspase-3 and -9.

Didymin enhanced RKIP expression

RKIP has been shown to be involved in several cell signaling cascades [14]. In the present study, PDGF-BB-stimulated cells showed higher level of RKIP than that of normal control cells. Compared to the PDGF-BB group, didymin treatment led to significant increase in the RKIP expression in both the liver tissues (Fig. 4A) and HSC-T6 cells (Fig. 4B), but this effect was largely abolished by locostatin.

Fig. 4

Effects of didymin on RKIP expression (A-B), ERK/MAPK (C-F) and PI3K/Akt (G-H) pathways. In the illustration A, the bands I to V represented the normal control group, didymin control group, CCl4 model group, CD group and CDL group, respectively. aP<0.05 vs. the CCl4 model group; bP<0.05 vs. the CDL group. In the illustrations B to H, the bands 1 to 4 represented the normal control group, PDGF-BB group, DP group and DLP group. *P<0.05 vs. normal control group, #P<0.05 vs. PDGF-BB group and P<0.05 vs. DLP group.

Fig. 4

Effects of didymin on RKIP expression (A-B), ERK/MAPK (C-F) and PI3K/Akt (G-H) pathways. In the illustration A, the bands I to V represented the normal control group, didymin control group, CCl4 model group, CD group and CDL group, respectively. aP<0.05 vs. the CCl4 model group; bP<0.05 vs. the CDL group. In the illustrations B to H, the bands 1 to 4 represented the normal control group, PDGF-BB group, DP group and DLP group. *P<0.05 vs. normal control group, #P<0.05 vs. PDGF-BB group and P<0.05 vs. DLP group.

Close modal

Didymin inhibited the ERK/MAPK pathway

To investigate the underlying mechanism of didymin on HSC-T6 cell proliferation and apoptosis, we examined the effect of didymin on the key intracellular ERK/MAPK pathway. Compared to the normal control group, PDGF-BB administration significantly increased the phosphorylations of MEK and ERK (Fig. 4 C and D), while decreased the phosphorylations of p38 and JNK (Fig. 4 E and F). However, didymin treatment notably reversed these effects induced by PDGF-BB. Interestingly, the effect of didymin on ERK/MAPK pathway was greatly abolished by locostatin.

Didymin inhibited the PI3K/Akt pathway

The PI3K signaling cascade regulates cell proliferation, survival, differentiation and apoptosis [15]. To fully understand the protective effect of didymin on HSC-T6 cell proliferation and apoptosis, we further investigated the effects of didymin on PI3K/Akt pathway. The results showed that the expression of PI3K and the phosphorylations of Akt in the PDGF-BB group were higher than those of the normal control group. Compared to the PDGF-BB group, didymin treatment significantly decreased the expressions of PI3K (Fig. 4G) and the phosphorylations of Akt (Fig. 4H). However, these effects were partially reversed by locostatin.

In the present study, hepatic fibrosis model was induced by CCl4 in rats. We found that CCl4 caused severe histological changes to the liver. But, these changes were significantly attenuated by didymin treatment. Additionally, our study revealed a significant increase in the activities of serum ALT and AST and the level of inflammatory cytokine TNF-α when exposed to CCl4, indicating serious hepatocellular injury. Didymin treatment significantly decreased the activities of both the serum enzymes and the level of TNF-α. Furthermore, CCl4 led to extensive accumulation of collagen in the liver tissues and significant increase in the contents of the collagen-related indicators including hepatic Hyp, PCIII, LN and HA. However, didymin treatment notably reversed these abnormal changes of collagen induced by CCl4. These results indicate that didymin can alleviate liver injury and fibrosis induced by CCl4.

HSCs play a critical role in the development of hepatic fibrosis. To fully understand the underlying mechanism of didymin against hepatic fibrosis, we further investigated the effect of didymin on HSCs biological behaviors including proliferation, apoptosis, cell cycle, mitochondrial function and the key intracellular signaling pathways such as ERK/MAPK and PI3K/Akt. As it is well known, apoptosis plays an important role in cells proliferation, differentiation, senescence and death. The potential to induce apoptosis has become an important topic in the study of anti-tumor drugs [16]. In the present study, didymin treatment decreased HSC-T6 cell proliferation in a dose-and time-dependent manner. Flow cytometric analysis showed that it significantly induced HSC-T6 cell apoptosis. These results suggest that didymin exerts anti-hepatic fibrosis mainly through inhibiting HSCs vitality and promoting cell apoptosis.

Cell cycle is regulated by a series of positive and negative mediators that act at sequential points throughout the cell cycle. We found that didymin treatment significantly increased the percentage of cells in G2 phase and inhibited the cell cycle-related regulators including cyclin B1, cyclin D1 and CDK4. Ii is well know that Cyclin D1 is a key G2/M phase regulator. The increase of cyclin D1 during G2 phase leads to progression to mitosis [17]. Thus, didymin induced cell cycle arrest in at G2 phase, likely through the inhibition of cyclin D1. In addition, we observed a decrease in the level of CDK4 commonly associated with the G1 transition and G2/M phase arrest. Cyclin B1 is a key regulatory protein controlling cell cycle progression in vertebrates. Depletion of cyclin B1 disrupts the spindle checkpoint functions and chromosomes alignment during mitosis [18]. In this context, the ability of didymin to down-regulate cyclin B1 in HSC-T6 cells represents a potentially significant mechanism of action for its anti-proliferative effects in HSC-T6 cells.

Mitochondria play an important role in regulating apoptosis. Mitochondrial dysfunction causes the collapse of mitochondria membrane potential (MMP), which results in mitochondrial permeability transition pore (MPTP) opening, enabling cytochrome c release from mitochondria into the cytosol [19]. Bcl-2 family proteins, including pro-apoptotic proteins (such as Bax) and anti-apoptotic proteins (such as Bcl-2), tightly regulate the mitochondria-dependent pathway [20]. In the present study, didymin treatment caused significant loss of MMP in HSC-T6 cells and obvious translocation of cytochrome c from the mitochondria to the cytosol. Furthermore, didymin treatment significantly decreased Bcl-2 expression and increased Bax level. These results suggest that the mitochondrial pathway of cell death, including Bcl-2 family and cytochrome c, may be involved in didymin-induced HSC-T6 cell apoptosis.

In general, cytochrome c releases into the cytosol and triggers caspase-9 activation. Subsequently, this initiator caspase activates caspase-3, which is a critical executioner of apoptosis. Following caspase activation, an increasing number of cellular substrates and the DNA repair protein poly (ADP-ribose) polymerase (PARP) are degraded or cleaved, resulting in cell death [21]. In the present study, didymin treatment significantly increased the levels of caspase-3 and −9, which suggests that the caspase-dependent mitochondrial signaling pathway is also involved in didymin-induced cell apoptosis.

The MAPK signaling pathway is highly conserved and is involved in cell growth, differentiation, survival, and invasion [22]. The MAPK family includes extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and P38 kinase. ERK1/2 are key transducers of proliferation signals and are often activated by mitogens. In contrast, SAPK/JNK and p38 are poorly stimulated by mitogens but strongly activated by cellular stress. ERK and P38 have been recently reported to be involved in cancer cell death induced by anti-cancer reagents [23]. Once activated, ERK plays an important role in anti-apoptotic activity, while JNK and p38 are important for pro-apoptotic activity [24]. Our results showed that didymin treatment decreased the phosphorylations of MEK and ERK, but increased the phosphorylations of JNK and p38, suggesting that the MAPK signaling pathway is involved in didymin-induced apoptosis.

Additionally, PI3K/Akt inhibition by didymin may also contribute to its inhibitory effect on cell growth. PI3K/Akt is an important signaling pathway in regulating cell proliferation, growth, apoptosis, survival and metabolism by phosphorylating several substrates. The inhibition of Akt phosphorylation has been suggested as a novel target for therapeutic cancer agents [25]. Akt activation by PI3K reduces apoptosis and promotes tumor cell growth through phosphorylation to inactivate a variety of downstreamtargets [26]. Our results showed that didymin treatment significantly decreased PI3K expression and Akt phosphorylation, suggesting that didymin induced apoptosis, at least in part, by inhibiting the PI3K/Akt pathway.

Raf kinase inhibitor protein (RKIP) is a specific MAPK signaling pathway inhibitor. It directly interacts with both Raf-1 and MEK and disrupts the Raf-1/MEK interaction, thereby preventing MEK activation and its downstream targets [27]. Interestingly, didymin has been reported to partially exert its pro-apoptosis effects in neuroblastoma by up-regulating RKIP expression [7]. Therefore, to investigate whether the target of didymin against hepatic fibrosis was RKIP, locostatin was used to interfere with RKIP expression and the antagonism of didymin was analyzed in vivo and in vitro. Our results showed that didymin significantly increased RKIP expression both in the liver tissues and HSC-T6 cells. Importantly, the protective effects of didymin on liver injury and collagen deposition in rats with hepatic fibrosis were greatly abolished by the specific RKIP inhibitor locostatin. The cell experiments in vitro showed that locostatin revealed partial reversal of didymin-induced inhibitions of cell proliferation. The promotive effect of didymin on cell apoptosis was also significantly inhibited by locostatin. Moreover, locostatin caused a partial reversal in didymin-induced ERK/MAPK and PI3K/Akt signaling pathways inactivation. These contradictive findings suggest that RKIP up-regulation represents a potential mechanism of action of didymin, specifically for its anti-fibrosis effects.

In summary, our studies showed that didymin treatment significantly alleviated liver injury and collagen production induced by CCl4 in rats. It notably inhibited HSC-T6 cell proliferation, predominantly by inducing apoptosis mediated by disrupting the cell cycle distribution, inducing mitochondrial dysfunction, regulating Bcl-2 family proteins and activating caspase-3/9. Moreover, didymin enhanced RKIP expression, thereby resulting in the inhibition of the ERK/MAPK and PI3K/Akt signaling pathways, which also contributed to didymin-induced apoptosis. These results indicate that didymin is likely to have potential as a new chemotherapeutic agent for the treatment of liver injury and fibrosis.

The authors gratefully acknowledge the financial support provided by the National Natural Science Foundation of China (No. 81260505; No.81473431) and the Guangxi Natural Science Foundation (2014GXNSFAA118155; 2014GXNSFAA118154).

The authors declare that there are no conflicts of interest.

1.
Hetherington AM, Sawyez CG, Zilberman E, Stoianov AM, Robson DL, Borradaile NM: Differential lipotoxic effects of palmitate and oleate in activated human hepatic stellate cells and epithelial hepatoma cells. Cell Physiol Biochem 2016;39:1648-1662.
2.
Kim MJ, Park SA, Kim CH, Park SY, Kim JS, Kim DK, Nam JS, Sheen YY: Tgf-beta type i receptor kinase inhibitor ew-7197 suppresses cholestatic liver fibrosis by inhibiting hif1alpha-induced epithelial mesenchymal transition. Cell Physiol Biochem 2016;38:571-588.
3.
Bataller R, Brenner DA: Liver fibrosis. J Clin Invest 2005;115:209-218.
4.
Zhang X, Tan Z, Wang Y, Tang J, Jiang R, Hou J, Zhuo H, Wang X, Ji J, Qin X, Sun B: Ptpro-associated hepatic stellate cell activation plays a critical role in liver fibrosis. Cell Physiol Biochem 2015;35:885-898.
5.
Bohanon FJ, Wang X, Ding C, Ding Y, Radhakrishnan GL, Rastellini C, Zhou J, Radhakrishnan RS: Oridonin inhibits hepatic stellate cell proliferation and fibrogenesis. J Surg Res 2014;190:55-63.
6.
Hung J, Hsu Y, Ko Y, Tsai Y, Yang C, Huang M, Kuo P: Didymin, a dietary flavonoid glycoside from citrus fruits, induces fas-mediated apoptotic pathway in human non-small-cell lung cancer cells in vitro and in vivo. Lung Cancer 2010;68:366-374.
7.
Singhal J, Nagaprashantha LD, Vatsyayan R, Awasthi S, Singhal SS: Didymin induces apoptosis by inhibiting n-myc and upregulating rkip in neuroblastoma. Cancer Prev Res 2012;5:473-483.
8.
Zhu S, Mc Henry KT, Lane WS, Fenteany G: A chemical inhibitor reveals the role of raf kinase inhibitor protein in cell migration. Chem Biol 2005;12:981-991.
9.
Zhang J, Li L, Liu X, Wang Y, Zhao D: Study on chemical constituents of artemisia sphaerocephala. Zhongguo Zhong Yao Za Zhi 2012;37:238-242.
10.
Patsenker E, Popov Y, Wiesner M, Goodman SL, Schuppan D: Pharmacological inhibition of the vitronectin receptor abrogates pdgf-bb-induced hepatic stellate cell migration and activation in vitro. J Hepatol 2007;46:878-887.
11.
Ma J, Li F, Liu L, Cui D, Wu X, Jiang X, Jiang H: Raf kinase inhibitor protein inhibits cell proliferation but promotes cell migration in rat hepatic stellate cells. Liver International 2009;29:567-574
12.
Reed JC: Bcl-2 family proteins; in Hickman JA and Dive C (ed): Apoptosis and cancer chemotherapy. Manchester, Springer, 1999, pp 99-116.
13.
Graña X, Reddy EP: Cell cycle control in mammalian cells: Role of cyclins, cyclin dependent kinases (cdks), growth suppressor genes and cyclin-dependent kinase inhibitors (ckis). Oncogene 1995;11:211-219.
14.
Keller ET, Fu Z, Brennan M: The role of raf kinase inhibitor protein (rkip) in health and disease. Biochem Pharmacol 2004;68:1049-1053.
15.
Vanhaesebroeck B, Guillermet-Guibert J, Graupera M, Bilanges B: The emerging mechanisms of isoform-specific pi3k signalling. Nat Rev Mol Cell Bio 2010;11:329-341.
16.
Krysko DV, Vanden Berghe T, D'Herde K, Vandenabeele P: Apoptosis and necrosis: Detection, discrimination and phagocytosis. Methods 2008;44:205-221.
17.
Guo Y, Stacey DW, Hitomi M: Post-transcriptional regulation of cyclin d1 expression during g2 phase. Oncogene 2002;21:7545-7556.
18.
Chen Q, Zhang X, Jiang Q, Clarke PR, Zhang C: Cyclin b1 is localized to unattached kinetochores and contributes to efficient microtubule attachment and proper chromosome alignment during mitosis. Cell Res 2008;18:268-280.
19.
Wu W, Zhou X, Liu P, Fei W, Li L, Yun H: Isoflurane reduces hypoxia/reoxygenation-induced apoptosis and mitochondrial permeability transition in rat primary cultured cardiocytes. BMC Anesthesiol 2014:14-17.
20.
Kong D, Zheng T, Zhang M, Wang D, Du S, Li X, Fang J, Cao X: Static mechanical stress induces apoptosis in rat endplate chondrocytes through mapk and mitochondria-dependent caspase activation signaling pathways. PLoS One 2013;8:e69403.
21.
Singh N, Sarkar J, Sashidhara KV, Ali S, Sinha S: Anti-tumour activity of a novel coumarin-chalcone hybrid is mediated through intrinsic apoptotic pathway by inducing puma and altering bax/bcl-2 ratio. Apoptosis 2014;19:1017-1028.
22.
O'Neill E, Kolch W: Conferring specificity on the ubiquitous raf/mek signalling pathway. Br J Cancer 2004;90:283-288.
23.
Chen L, He H, Li H, Zheng J, Heng W, You JF, Fang W: Erk1/2 and p38 pathways are required for p2y receptor-mediated prostate cancer invasion. Cancer Lett 2004;215:239-247.
24.
Sheth K, Friel J, Nolan B, Bankey P: Inhibition of p38 mitogen activated protein kinase increases lipopolysaccharide induced inhibition of apoptosis in neutrophils by activating extracellular signal-regulated kinase. Surgery 2001;130:242-248.
25.
Fresno Vara JÁ, Casado E, de Castro J, Cejas P, Belda-Iniesta C, González-Barón M: Pi3k/akt signalling pathway and cancer. Cancer Treat Rev 2004;30:193-204.
26.
Franke TF, Hornik CP, Segev L, Shostak GA, Sugimoto C: Pi3k/akt and apoptosis: Size matters. Oncogene 2003;22:8983-8998.
27.
Yeung K, Janosch P, McFerran B, Rose DW, Mischak H, Sedivy JM, Kolch W: Mechanism of suppression of the raf/mek/extracellular signal-regulated kinase pathway by the raf kinase inhibitor protein. Mol Cell Biol 2000;20:3079-3085.
Open Access License / Drug Dosage / Disclaimer
This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.