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Silymarin Accelerates Liver Regeneration after Partial Hepatectomy.

Wu JP, Tsai CC, Yeh YL, Lin YM, Lin CC, Day CH, Shen CY, Padma VV, Pan LF, Huang CY - Evid Based Complement Alternat Med (2015)

Bottom Line: Western blot and RT-PCR were conducted to detect the cell cycle activities and silymarin effects on hepatic regeneration.Silymarin led to increased G1 phase (cyclin D1/pRb), S phase (cyclin E/E2F), G2 phase (cyclin B), and M phase (cyclin A) protein and mRNA at 6 hrs, 24 hrs, and 72 hrs PHx.HGF, TGFα, and TGFβ1 growth factor expressions were also enhanced.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan.

ABSTRACT
Partial hepatectomy (PHx) is a liver regeneration physiological response induced to maintain homeostasis. Liver regeneration evolved presumably to protect wild animals from catastrophic liver loss caused by toxins or tissue injury. Silymarin (Sm) ability to stimulate liver regeneration has been an object of curiosity for many years. Silymarin has been investigated for use as an antioxidant and anticarcinogen. However, its use as a supportive treatment for liver damage is elusive. In this study, we fed silymarin (Sm, 25 mg/kg) to male Sprague-Dawley rats for 7 weeks. Surgical 2/3 PHx was then conducted on the rats at 6 hrs, 24 hrs, and 72 hrs. Western blot and RT-PCR were conducted to detect the cell cycle activities and silymarin effects on hepatic regeneration. The results showed that silymarin enhanced liver regeneration by accelerating the cell cycle in PHx liver. Silymarin led to increased G1 phase (cyclin D1/pRb), S phase (cyclin E/E2F), G2 phase (cyclin B), and M phase (cyclin A) protein and mRNA at 6 hrs, 24 hrs, and 72 hrs PHx. HGF, TGFα, and TGFβ1 growth factor expressions were also enhanced. We suggest that silymarin plays a crucial role in accelerated liver regeneration after PHx.

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Partial hepatectomy is a delayed cell cycle physiological response. (a) Quantification of western blot densitometry analysis of G1 phase checkpoint proteins, cyclin D1 and pRb, expression levels in Sham, PHx, and silymarin (Sm) treatment partial hepatectomy at 6 hrs, 24 hrs, and 72 hrs. All data are presented as means ± SEM, ∗p < 0.05, as compared with the corresponding sham group. #p < 0.05, ##p < 0.01, as compared with the corresponding PHx. ap < 0.05 as compared with 6 hrs sham group. bp < 0.05 as compared with 24 hrs sham group. cp < 0.05 as compared with 6 hrs PHx group. dp < 0.05 as compared with 24 hrs PHx group. (b) Quantification of western blot densitometry analysis of S phase checkpoint proteins, cyclin E and E2F, expression levels in Sham, PHx, and silymarin (Sm) treatment partial hepatectomy at 6 hrs, 24 hrs, and 72 hrs. All data are presented as means ± SEM; ∗p < 0.05, ∗∗p < 0.01 as compared with the corresponding sham group. #p < 0.05, as compared with the corresponding PHx. ap < 0.05, aap < 0.01 as compared with 6 hrs sham group. bp < 0.05 as compared with 24 hrs sham group. cp < 0.05, ccp < 0.01, as compared with 6 hrs PHx group. dp < 0.05, ddp < 0.01 as compared with 24 hrs PHx group. (c) Representative cell cycle in sham and PHx at 6 hrs, 24 hrs, and 72 hrs three different times.
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fig2: Partial hepatectomy is a delayed cell cycle physiological response. (a) Quantification of western blot densitometry analysis of G1 phase checkpoint proteins, cyclin D1 and pRb, expression levels in Sham, PHx, and silymarin (Sm) treatment partial hepatectomy at 6 hrs, 24 hrs, and 72 hrs. All data are presented as means ± SEM, ∗p < 0.05, as compared with the corresponding sham group. #p < 0.05, ##p < 0.01, as compared with the corresponding PHx. ap < 0.05 as compared with 6 hrs sham group. bp < 0.05 as compared with 24 hrs sham group. cp < 0.05 as compared with 6 hrs PHx group. dp < 0.05 as compared with 24 hrs PHx group. (b) Quantification of western blot densitometry analysis of S phase checkpoint proteins, cyclin E and E2F, expression levels in Sham, PHx, and silymarin (Sm) treatment partial hepatectomy at 6 hrs, 24 hrs, and 72 hrs. All data are presented as means ± SEM; ∗p < 0.05, ∗∗p < 0.01 as compared with the corresponding sham group. #p < 0.05, as compared with the corresponding PHx. ap < 0.05, aap < 0.01 as compared with 6 hrs sham group. bp < 0.05 as compared with 24 hrs sham group. cp < 0.05, ccp < 0.01, as compared with 6 hrs PHx group. dp < 0.05, ddp < 0.01 as compared with 24 hrs PHx group. (c) Representative cell cycle in sham and PHx at 6 hrs, 24 hrs, and 72 hrs three different times.

Mentions: During regeneration after 70% hepatectomy, the liver was divided one or two times and began to regenerate and then return to quiescence. We know that liver regeneration is a physiological response induced for maintaining homeostasis. According to Table 1, the result showed us the postoperative liver weight (g) which increased from 3.7 ± 0.67 (6 hrs PHx) to 5.3 ± 0.36 (24 hrs PHx) (p < 0.001 versus 6 hrs PHx), to 8.7 ± 1.71 (72 hrs PHx) (p < 0.0001 versus 6 hrs PHx), and to 10.8 ± 0.62 (168 hrs PHx) (p < 0.0001 versus 6 hrs PHx). Partial liver weight (g) at 72 hrs PHx has significantly increased but decreased at 168 hrs. In turn, the remnant liver weight (g) also decreased at 72 hrs PHx, from 4.7 ± 0.99 (at 6 hrs PHx) to 2.8 ± 0.64 (at 72 hrs PHx, p < 0.001 versus at 6 hrs PHx), but with no significant difference at 168 hrs. Thus, the liver regeneration (%) increased from 10.62% at 24 hrs to 76.32% at 72 hrs and from the highest to 119.55% at 168 hrs PHx. Given the above evidence, liver regeneration is time dependent. Partial hepatectomy (PHx) is a complex physiological response that takes place after the loss of hepatocytes caused by viral injury or secondary liver resection. During liver regeneration, a series of resections take place to maintain homeostasis to restore normal hepatic mass and structure. Virtually, all of the surviving hepatocytes undergo cellular proliferation due to tissue remodeling. PHx is a delayed physiological response during liver regeneration. We want to determine whether silymarin will accelerate the cell cycle to return to normal conditions during liver regeneration. Therefore, we detected cell cycle check proteins, cyclin D1/pRb in G1 phase and cyclin E/E2F in S phase, in sham, PHx, and silymarin treatment with PHx by western blot analysis. After partial hepatectomy, liver regeneration began to proliferate and cell cycle prolonged. We found G1 phase extended, from 6 hrs to 24 hrs and into S phase at 72 hrs during liver regeneration. Therefore, we could find cyclin D1 and pRb protein expression increased at 6 hrs PHx, but not significantly in cyclin E and E2F, when compared with sham, respectively (∗p < 0.05 versus sham). Partial hepatectomy treatment with silymarin has strongly enhanced cyclin D1, pRb, cyclin E, and E2F protein expression levels (∗p < 0.05 versus sham, #p < 0.05 versus PHx) (Figure 1(a)). After 24 hrs of liver regeneration, we found the strongest regeneration; cyclin D1, pRb, cyclin E, and E2F protein expression all were increased after PHx. We also could find silymarin improved this stage (∗p < 0.05 versus sham, #p < 0.05 versus PHx) (Figure 1(b)). On the other hand, during long term 72 hrs PHx, cyclin D1, and pRb protein expression were decreased compared with sham, respectively. Silymarin also was induced (∗p < 0.05 versus sham, ##p < 0.05 versus PHx). In turn, cyclin E and E2F had increased. During this time, silymarin may has been losing functions to improve cell cycle, when compared with PHx (##p < 0.05 versus PHx); however, compared with the sham, silymarin also increased (∗p < 0.05 versus sham) (Figure 1(c)). After hepatic growth and restructuring, DNA synthesis was completed by 72 hrs and liver regeneration eventually stops. We focused on liver regeneration initiation and compared termination with it. Thus, we examined the cell cycle check point protein at different times. We examined cyclin D1/pRb in G1 phase and cyclin E/E2F in S phase protein expression by western blot analysis (Figures 2(a) and 2(b)). We found cyclin D1 was decreased at 24 hrs sham but increased at 72 hrs (ap < 0.05 versus 6 hrs sham, bp < 0.05 versus 24 hrs sham). When compared with PHx at 6 hrs, 24 hrs, and 72 hrs, we found cyclin D1 at 24 hrs PHx was increased (cp < 0.05 versus 6 hrs PHx) but at 72 hrs PHx was decreased (cp < 0.05 versus 6 hrs PHx, dp < 0.05 versus 24 hrs PHx). However, pRb protein expression levels were increased only at 72 hrs sham (ap < 0.05 versus 6 hrs sham, bp < 0.05 versus 24 hrs sham). However, after 24 hrs PHx, pRb protein expression was increased (cp < 0.05 versus 6 hrs PHx). After 72 hrs PHx, pRb was decreased (dp < 0.05 versus 24 hrs PHx). No significant difference compared with 6 hrs PHx was shown. Notwithstanding, silymarin also enhanced cyclin D1/pRb protein expression at all the three PHx times, 6 hrs, 24 hrs, and 72 hrs PHx, respectively (∗p < 0.05 versus sham, #p < 0.05, ##p < 0.01 versus PHx) (Figures 1 and 2(a)). On the other hand, in cell cycle S phase, we found cyclin E was increased at 24 hrs sham (ap < 0.05 versus 6 hrs sham), but at 72 hrs sham has conversely to 6 hrs sham. After hepatectomy, we found cyclin E protein expression increased at 24 hrs PHx and more at 72 hrs PHx (cp < 0.05, ccp < 0.01 versus 6 hrs PHx, dp < 0.05 versus 24 hrs PHx) (Figure 2(b)). On the other hand, E2F increased expression at 24 hrs and 72 hrs sham compared with 6 hrs sham (ap < 0.05, aap < 0.01 versus 6 hrs sham, bp < 0.05 versus 24 hrs sham). When compared with 6 hrs PHx, we found E2F protein expression increased at 24 hrs PHx but at 72 hrs PHx has more than 6 hrs and 24 hrs PHx (cp < 0.05, ccp < 0.01 versus 6 hrs PHx; dp < 0.05, ddp < 0.01 versus 24 hrs PHx). However, silymarin also has strong functions to induce cyclin E/E2F protein expression after PHx (∗p < 0.05 versus sham, #p < 0.05, versus PHx) (Figures 1 and 2(b)). Therefore, we could determine silymarin stimulated cell cycle protein expression for entry into S phase. After hepatic growth and restructuring, DNA synthesis is mostly complete by 72 hrs and liver regeneration eventually stops. We focused on liver regeneration initiation and compared termination with it. Furthermore, we detected whether silymarin regulated the cell cycle and accelerated it to become normal. We examined G2 and M phase check point protein, cyclin B and cyclin A, protein, and mRNA expression using western and RT-PCR. It is generally believed that the external controls have three important control points in the cell cycle at the end of G2 phase (G2/M transition), in mitosis, and in G1 phase. Intrinsic control mechanisms ensure that the cycle is executed completely. Therefore, we determine whether silymarin also improved the cell cycle at the end of G2 phase. We used western blot analysis and RT-PCR to detect protein and mRNA expression in the sham and PHx to determine the cell cycle. The results showed that the G2 phase check point protein, cyclin B, increased at 24 hrs and 72 hrs in the sham (ap < 0.05 versus 6 hrs sham). However, cyclin B at 24 hrs in the PHx decreased compared with the sham at 24 and 6 hrs in the PHx, respectively. After 72 hrs in the PHx, cyclin B increased compared with the PHx at 24 hrs (dp < 0.05, versus 24 hrs PHx) (Figure 3(a)). Cyclin B mRNA also decreased at 24 hrs in the PHx but increased at 72 hrs in the PHx (Figures 3(b) and 3(c)). Silymarin presented significant effects at 6 hrs, 24 hrs, and 72 hrs in the PHx to improve G1 phase into M phase. We also detected cyclin A protein and mRNA in the mitosis phase. We found that protein and mRNA expression increased at 24 hrs and 72 hrs in the sham. When compared with the sham, we found that cyclin A protein decreased at 6 hrs and 24 hrs in the PHx, respectively (∗p < 0.05 versus sham). However, cyclin A mRNA decreased at 24 hrs in the PHx and increased at 72 hrs in the PHx, when compared with 6 hrs PHx (Figures 4(a) and 4(b)). Interestingly, during liver regeneration after PHx, we found cyclin A mRNA and protein expression increased at 72 hrs. PHx led cyclin A expression to delay to 72 hrs (Figure 4(c)). Interestingly, silymarin had no significant beneficial effect on cell cycle functions after 72 hrs PHx. To determine whether growth factors are the primary accelerated cell cycle effects, we examined liver regeneration related growth factors, HGF and TGFα, expression. We found that HGF protein and mRNA expression decreased at 6 hrs in the PHx, but this was not found at 72 hrs in the PHx. We also found that HGF increased after treatment with silymarin comparing the sham and PHx at 6 hrs or 72 hrs (Figures 5(a) and 5(b)). TGFβ1 seems to be inhibited hepatocyte DNA synthesis that is negative regulator of liver growth. In order to determine whether cell proliferation and cell cycle are coordinately regulated by TGFβ1 in regeneration, the result showed that TGFβ1 protein was not significantly different in sham rats. However, after hepatectomy TGFβ1 protein expression levels increased at 24 hrs PHx (cp < 0.05 versus 6 hrs PHx), with the greatest expression at 72 hrs PHx (ccp < 0.01 versus 6 hrs PHx, dp < 0.05, versus 24 hrs PHx) (Figure 6(a)). On the other hand, mRNA expression increased at 24 hrs and 72 hrs sham (Figure 6(b)). After partial hepatectomy, we also found mRNA expression increased at 24 hrs and 72 hrs PHx (cp < 0.05, ccp < 0.01 versus 6 hrs PHx; dp < 0.05 versus 24 hrs PHx). One possibility is that TGFβ1 is a mitoinhibitor that causes the end of regeneration. Therefore, this pointed out that liver regeneration proceeds to completion. Obviously, hepatocytes proceed through regeneration despite the TGFβ1 increase. A further possibility is TGFβ1 mitoinhibitory effects return by 96 hrs and hepatocyte proliferation stops between 48 and 72 hrs. Resistance to TGFβ1 by regenerating hepatocytes may allow hepatocytes to proliferate even through TGFβ1 concentrations increasing. Silymarin improved TGFβ1 protein and mRNA expression (∗p < 0.05, ∗∗p < 0.01 versus sham; #p < 0.01, ##p < 0.05 versus PHx) (Figure 6). Growth factors in the G1 phase have stronger expression. TGFα presented increased protein expression at 24 hrs in the PHx. This was not found at 6 and 72 hrs. After treatment with silymarin TGFα protein expression increased at 6 hrs, 24 hrs, and 72 hrs in the PHx. We used the Ki-67 antibody to examine immunofluorescence and Brdu antibodies using the immunohistochemistry assay to determine cell proliferation. The Ki-67 protein is present during all active phases in the cell cycle (G1, S, G2, and mitosis) but is absent from resting cells (G0). Therefore, both PHx and after PHx treatment Sm presented higher positive Ki-67 antibody expression from immunofluorescence analysis. Lower 10% was expressed as lower proliferation. PHx is a hyperplasia considered to be a physiological response to a specific stimulus. PHx and Sm-treated cells with hyperplastic growth remained subject to normal regulatory control mechanisms (Figure 7(a)) (green color). And Ki-67 staining is different and BrdU staining is detected only in the S phase of the cell, which is in the DNA replication phase of the cell. BrdU is a uridine derivative that will replace thymidine incorporated into the DNA of cells in S phase (Figure 7(b), yellow arrows). Brown color BrsU nuclear cells were observed at 24 hrs and 72 hrs PHx and Sm-treated cells. We suggest that silymarin could lead cell cycle accelerated and made delated cell cycle run fastly to feedback normal (Figure 8). During 72 hrs PHx, silymarin may deliver in the aggregate a set of signals that lead to regeneration termination in the cyclin A phase. Silymarin can function as a hepatoprotectant agent. In the future, silymarin may be useful as an adjuvant for the treatment of specific liver diseases.


Silymarin Accelerates Liver Regeneration after Partial Hepatectomy.

Wu JP, Tsai CC, Yeh YL, Lin YM, Lin CC, Day CH, Shen CY, Padma VV, Pan LF, Huang CY - Evid Based Complement Alternat Med (2015)

Partial hepatectomy is a delayed cell cycle physiological response. (a) Quantification of western blot densitometry analysis of G1 phase checkpoint proteins, cyclin D1 and pRb, expression levels in Sham, PHx, and silymarin (Sm) treatment partial hepatectomy at 6 hrs, 24 hrs, and 72 hrs. All data are presented as means ± SEM, ∗p < 0.05, as compared with the corresponding sham group. #p < 0.05, ##p < 0.01, as compared with the corresponding PHx. ap < 0.05 as compared with 6 hrs sham group. bp < 0.05 as compared with 24 hrs sham group. cp < 0.05 as compared with 6 hrs PHx group. dp < 0.05 as compared with 24 hrs PHx group. (b) Quantification of western blot densitometry analysis of S phase checkpoint proteins, cyclin E and E2F, expression levels in Sham, PHx, and silymarin (Sm) treatment partial hepatectomy at 6 hrs, 24 hrs, and 72 hrs. All data are presented as means ± SEM; ∗p < 0.05, ∗∗p < 0.01 as compared with the corresponding sham group. #p < 0.05, as compared with the corresponding PHx. ap < 0.05, aap < 0.01 as compared with 6 hrs sham group. bp < 0.05 as compared with 24 hrs sham group. cp < 0.05, ccp < 0.01, as compared with 6 hrs PHx group. dp < 0.05, ddp < 0.01 as compared with 24 hrs PHx group. (c) Representative cell cycle in sham and PHx at 6 hrs, 24 hrs, and 72 hrs three different times.
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fig2: Partial hepatectomy is a delayed cell cycle physiological response. (a) Quantification of western blot densitometry analysis of G1 phase checkpoint proteins, cyclin D1 and pRb, expression levels in Sham, PHx, and silymarin (Sm) treatment partial hepatectomy at 6 hrs, 24 hrs, and 72 hrs. All data are presented as means ± SEM, ∗p < 0.05, as compared with the corresponding sham group. #p < 0.05, ##p < 0.01, as compared with the corresponding PHx. ap < 0.05 as compared with 6 hrs sham group. bp < 0.05 as compared with 24 hrs sham group. cp < 0.05 as compared with 6 hrs PHx group. dp < 0.05 as compared with 24 hrs PHx group. (b) Quantification of western blot densitometry analysis of S phase checkpoint proteins, cyclin E and E2F, expression levels in Sham, PHx, and silymarin (Sm) treatment partial hepatectomy at 6 hrs, 24 hrs, and 72 hrs. All data are presented as means ± SEM; ∗p < 0.05, ∗∗p < 0.01 as compared with the corresponding sham group. #p < 0.05, as compared with the corresponding PHx. ap < 0.05, aap < 0.01 as compared with 6 hrs sham group. bp < 0.05 as compared with 24 hrs sham group. cp < 0.05, ccp < 0.01, as compared with 6 hrs PHx group. dp < 0.05, ddp < 0.01 as compared with 24 hrs PHx group. (c) Representative cell cycle in sham and PHx at 6 hrs, 24 hrs, and 72 hrs three different times.
Mentions: During regeneration after 70% hepatectomy, the liver was divided one or two times and began to regenerate and then return to quiescence. We know that liver regeneration is a physiological response induced for maintaining homeostasis. According to Table 1, the result showed us the postoperative liver weight (g) which increased from 3.7 ± 0.67 (6 hrs PHx) to 5.3 ± 0.36 (24 hrs PHx) (p < 0.001 versus 6 hrs PHx), to 8.7 ± 1.71 (72 hrs PHx) (p < 0.0001 versus 6 hrs PHx), and to 10.8 ± 0.62 (168 hrs PHx) (p < 0.0001 versus 6 hrs PHx). Partial liver weight (g) at 72 hrs PHx has significantly increased but decreased at 168 hrs. In turn, the remnant liver weight (g) also decreased at 72 hrs PHx, from 4.7 ± 0.99 (at 6 hrs PHx) to 2.8 ± 0.64 (at 72 hrs PHx, p < 0.001 versus at 6 hrs PHx), but with no significant difference at 168 hrs. Thus, the liver regeneration (%) increased from 10.62% at 24 hrs to 76.32% at 72 hrs and from the highest to 119.55% at 168 hrs PHx. Given the above evidence, liver regeneration is time dependent. Partial hepatectomy (PHx) is a complex physiological response that takes place after the loss of hepatocytes caused by viral injury or secondary liver resection. During liver regeneration, a series of resections take place to maintain homeostasis to restore normal hepatic mass and structure. Virtually, all of the surviving hepatocytes undergo cellular proliferation due to tissue remodeling. PHx is a delayed physiological response during liver regeneration. We want to determine whether silymarin will accelerate the cell cycle to return to normal conditions during liver regeneration. Therefore, we detected cell cycle check proteins, cyclin D1/pRb in G1 phase and cyclin E/E2F in S phase, in sham, PHx, and silymarin treatment with PHx by western blot analysis. After partial hepatectomy, liver regeneration began to proliferate and cell cycle prolonged. We found G1 phase extended, from 6 hrs to 24 hrs and into S phase at 72 hrs during liver regeneration. Therefore, we could find cyclin D1 and pRb protein expression increased at 6 hrs PHx, but not significantly in cyclin E and E2F, when compared with sham, respectively (∗p < 0.05 versus sham). Partial hepatectomy treatment with silymarin has strongly enhanced cyclin D1, pRb, cyclin E, and E2F protein expression levels (∗p < 0.05 versus sham, #p < 0.05 versus PHx) (Figure 1(a)). After 24 hrs of liver regeneration, we found the strongest regeneration; cyclin D1, pRb, cyclin E, and E2F protein expression all were increased after PHx. We also could find silymarin improved this stage (∗p < 0.05 versus sham, #p < 0.05 versus PHx) (Figure 1(b)). On the other hand, during long term 72 hrs PHx, cyclin D1, and pRb protein expression were decreased compared with sham, respectively. Silymarin also was induced (∗p < 0.05 versus sham, ##p < 0.05 versus PHx). In turn, cyclin E and E2F had increased. During this time, silymarin may has been losing functions to improve cell cycle, when compared with PHx (##p < 0.05 versus PHx); however, compared with the sham, silymarin also increased (∗p < 0.05 versus sham) (Figure 1(c)). After hepatic growth and restructuring, DNA synthesis was completed by 72 hrs and liver regeneration eventually stops. We focused on liver regeneration initiation and compared termination with it. Thus, we examined the cell cycle check point protein at different times. We examined cyclin D1/pRb in G1 phase and cyclin E/E2F in S phase protein expression by western blot analysis (Figures 2(a) and 2(b)). We found cyclin D1 was decreased at 24 hrs sham but increased at 72 hrs (ap < 0.05 versus 6 hrs sham, bp < 0.05 versus 24 hrs sham). When compared with PHx at 6 hrs, 24 hrs, and 72 hrs, we found cyclin D1 at 24 hrs PHx was increased (cp < 0.05 versus 6 hrs PHx) but at 72 hrs PHx was decreased (cp < 0.05 versus 6 hrs PHx, dp < 0.05 versus 24 hrs PHx). However, pRb protein expression levels were increased only at 72 hrs sham (ap < 0.05 versus 6 hrs sham, bp < 0.05 versus 24 hrs sham). However, after 24 hrs PHx, pRb protein expression was increased (cp < 0.05 versus 6 hrs PHx). After 72 hrs PHx, pRb was decreased (dp < 0.05 versus 24 hrs PHx). No significant difference compared with 6 hrs PHx was shown. Notwithstanding, silymarin also enhanced cyclin D1/pRb protein expression at all the three PHx times, 6 hrs, 24 hrs, and 72 hrs PHx, respectively (∗p < 0.05 versus sham, #p < 0.05, ##p < 0.01 versus PHx) (Figures 1 and 2(a)). On the other hand, in cell cycle S phase, we found cyclin E was increased at 24 hrs sham (ap < 0.05 versus 6 hrs sham), but at 72 hrs sham has conversely to 6 hrs sham. After hepatectomy, we found cyclin E protein expression increased at 24 hrs PHx and more at 72 hrs PHx (cp < 0.05, ccp < 0.01 versus 6 hrs PHx, dp < 0.05 versus 24 hrs PHx) (Figure 2(b)). On the other hand, E2F increased expression at 24 hrs and 72 hrs sham compared with 6 hrs sham (ap < 0.05, aap < 0.01 versus 6 hrs sham, bp < 0.05 versus 24 hrs sham). When compared with 6 hrs PHx, we found E2F protein expression increased at 24 hrs PHx but at 72 hrs PHx has more than 6 hrs and 24 hrs PHx (cp < 0.05, ccp < 0.01 versus 6 hrs PHx; dp < 0.05, ddp < 0.01 versus 24 hrs PHx). However, silymarin also has strong functions to induce cyclin E/E2F protein expression after PHx (∗p < 0.05 versus sham, #p < 0.05, versus PHx) (Figures 1 and 2(b)). Therefore, we could determine silymarin stimulated cell cycle protein expression for entry into S phase. After hepatic growth and restructuring, DNA synthesis is mostly complete by 72 hrs and liver regeneration eventually stops. We focused on liver regeneration initiation and compared termination with it. Furthermore, we detected whether silymarin regulated the cell cycle and accelerated it to become normal. We examined G2 and M phase check point protein, cyclin B and cyclin A, protein, and mRNA expression using western and RT-PCR. It is generally believed that the external controls have three important control points in the cell cycle at the end of G2 phase (G2/M transition), in mitosis, and in G1 phase. Intrinsic control mechanisms ensure that the cycle is executed completely. Therefore, we determine whether silymarin also improved the cell cycle at the end of G2 phase. We used western blot analysis and RT-PCR to detect protein and mRNA expression in the sham and PHx to determine the cell cycle. The results showed that the G2 phase check point protein, cyclin B, increased at 24 hrs and 72 hrs in the sham (ap < 0.05 versus 6 hrs sham). However, cyclin B at 24 hrs in the PHx decreased compared with the sham at 24 and 6 hrs in the PHx, respectively. After 72 hrs in the PHx, cyclin B increased compared with the PHx at 24 hrs (dp < 0.05, versus 24 hrs PHx) (Figure 3(a)). Cyclin B mRNA also decreased at 24 hrs in the PHx but increased at 72 hrs in the PHx (Figures 3(b) and 3(c)). Silymarin presented significant effects at 6 hrs, 24 hrs, and 72 hrs in the PHx to improve G1 phase into M phase. We also detected cyclin A protein and mRNA in the mitosis phase. We found that protein and mRNA expression increased at 24 hrs and 72 hrs in the sham. When compared with the sham, we found that cyclin A protein decreased at 6 hrs and 24 hrs in the PHx, respectively (∗p < 0.05 versus sham). However, cyclin A mRNA decreased at 24 hrs in the PHx and increased at 72 hrs in the PHx, when compared with 6 hrs PHx (Figures 4(a) and 4(b)). Interestingly, during liver regeneration after PHx, we found cyclin A mRNA and protein expression increased at 72 hrs. PHx led cyclin A expression to delay to 72 hrs (Figure 4(c)). Interestingly, silymarin had no significant beneficial effect on cell cycle functions after 72 hrs PHx. To determine whether growth factors are the primary accelerated cell cycle effects, we examined liver regeneration related growth factors, HGF and TGFα, expression. We found that HGF protein and mRNA expression decreased at 6 hrs in the PHx, but this was not found at 72 hrs in the PHx. We also found that HGF increased after treatment with silymarin comparing the sham and PHx at 6 hrs or 72 hrs (Figures 5(a) and 5(b)). TGFβ1 seems to be inhibited hepatocyte DNA synthesis that is negative regulator of liver growth. In order to determine whether cell proliferation and cell cycle are coordinately regulated by TGFβ1 in regeneration, the result showed that TGFβ1 protein was not significantly different in sham rats. However, after hepatectomy TGFβ1 protein expression levels increased at 24 hrs PHx (cp < 0.05 versus 6 hrs PHx), with the greatest expression at 72 hrs PHx (ccp < 0.01 versus 6 hrs PHx, dp < 0.05, versus 24 hrs PHx) (Figure 6(a)). On the other hand, mRNA expression increased at 24 hrs and 72 hrs sham (Figure 6(b)). After partial hepatectomy, we also found mRNA expression increased at 24 hrs and 72 hrs PHx (cp < 0.05, ccp < 0.01 versus 6 hrs PHx; dp < 0.05 versus 24 hrs PHx). One possibility is that TGFβ1 is a mitoinhibitor that causes the end of regeneration. Therefore, this pointed out that liver regeneration proceeds to completion. Obviously, hepatocytes proceed through regeneration despite the TGFβ1 increase. A further possibility is TGFβ1 mitoinhibitory effects return by 96 hrs and hepatocyte proliferation stops between 48 and 72 hrs. Resistance to TGFβ1 by regenerating hepatocytes may allow hepatocytes to proliferate even through TGFβ1 concentrations increasing. Silymarin improved TGFβ1 protein and mRNA expression (∗p < 0.05, ∗∗p < 0.01 versus sham; #p < 0.01, ##p < 0.05 versus PHx) (Figure 6). Growth factors in the G1 phase have stronger expression. TGFα presented increased protein expression at 24 hrs in the PHx. This was not found at 6 and 72 hrs. After treatment with silymarin TGFα protein expression increased at 6 hrs, 24 hrs, and 72 hrs in the PHx. We used the Ki-67 antibody to examine immunofluorescence and Brdu antibodies using the immunohistochemistry assay to determine cell proliferation. The Ki-67 protein is present during all active phases in the cell cycle (G1, S, G2, and mitosis) but is absent from resting cells (G0). Therefore, both PHx and after PHx treatment Sm presented higher positive Ki-67 antibody expression from immunofluorescence analysis. Lower 10% was expressed as lower proliferation. PHx is a hyperplasia considered to be a physiological response to a specific stimulus. PHx and Sm-treated cells with hyperplastic growth remained subject to normal regulatory control mechanisms (Figure 7(a)) (green color). And Ki-67 staining is different and BrdU staining is detected only in the S phase of the cell, which is in the DNA replication phase of the cell. BrdU is a uridine derivative that will replace thymidine incorporated into the DNA of cells in S phase (Figure 7(b), yellow arrows). Brown color BrsU nuclear cells were observed at 24 hrs and 72 hrs PHx and Sm-treated cells. We suggest that silymarin could lead cell cycle accelerated and made delated cell cycle run fastly to feedback normal (Figure 8). During 72 hrs PHx, silymarin may deliver in the aggregate a set of signals that lead to regeneration termination in the cyclin A phase. Silymarin can function as a hepatoprotectant agent. In the future, silymarin may be useful as an adjuvant for the treatment of specific liver diseases.

Bottom Line: Western blot and RT-PCR were conducted to detect the cell cycle activities and silymarin effects on hepatic regeneration.Silymarin led to increased G1 phase (cyclin D1/pRb), S phase (cyclin E/E2F), G2 phase (cyclin B), and M phase (cyclin A) protein and mRNA at 6 hrs, 24 hrs, and 72 hrs PHx.HGF, TGFα, and TGFβ1 growth factor expressions were also enhanced.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan.

ABSTRACT
Partial hepatectomy (PHx) is a liver regeneration physiological response induced to maintain homeostasis. Liver regeneration evolved presumably to protect wild animals from catastrophic liver loss caused by toxins or tissue injury. Silymarin (Sm) ability to stimulate liver regeneration has been an object of curiosity for many years. Silymarin has been investigated for use as an antioxidant and anticarcinogen. However, its use as a supportive treatment for liver damage is elusive. In this study, we fed silymarin (Sm, 25 mg/kg) to male Sprague-Dawley rats for 7 weeks. Surgical 2/3 PHx was then conducted on the rats at 6 hrs, 24 hrs, and 72 hrs. Western blot and RT-PCR were conducted to detect the cell cycle activities and silymarin effects on hepatic regeneration. The results showed that silymarin enhanced liver regeneration by accelerating the cell cycle in PHx liver. Silymarin led to increased G1 phase (cyclin D1/pRb), S phase (cyclin E/E2F), G2 phase (cyclin B), and M phase (cyclin A) protein and mRNA at 6 hrs, 24 hrs, and 72 hrs PHx. HGF, TGFα, and TGFβ1 growth factor expressions were also enhanced. We suggest that silymarin plays a crucial role in accelerated liver regeneration after PHx.

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