Pharmaceutical Biology
ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20
Effect of earthworm active protein on fibroblast proliferation and its mechanism Shuliang Song, Yuling Wang, Kai Ji, Hao Liang & Aiguo Ji To cite this article: Shuliang Song, Yuling Wang, Kai Ji, Hao Liang & Aiguo Ji (2015): Effect of earthworm active protein on fibroblast proliferation and its mechanism, Pharmaceutical Biology, DOI: 10.3109/13880209.2015.1073333 To link to this article: http://dx.doi.org/10.3109/13880209.2015.1073333
Published online: 07 Oct 2015.
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Date: 22 November 2015, At: 18:41
PHARMACEUTICAL BIOLOGY 2015; EARLY ONLINE: 1–8 http://dx.doi.org/10.3109/13880209.2015.1073333
SHORT COMMUNICATION
Effect of earthworm active protein on fibroblast proliferation and its mechanism Shuliang Song1*, Yuling Wang1*, Kai Ji2, Hao Liang1, and Aiguo Ji1,3
Downloaded by [University of Otago] at 18:41 22 November 2015
1 Marine College Shandong University (weihai), Shandong, China, 2Plastic Surgery Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China, and 3School of Pharmaceutical Sciences Shandong University, Shandong, China
ABSTRACT
KEYWORDS
Context: Earthworms have been used as a traditional medicine in China from thousands of years. In recent years, research has demonstrated that earthworm extracts might promote wound healing; however, its mechanism is still unknown. Objective: The study investigates the mechanism and effects of earthworm active protein (EAP), on mouse embryonic fibroblast (NIH/3T3) proliferation. Materials and methods: The effects of earthworm active protein (EAP) in different concentrations (0, 25, 50, 100, 150, and 200 mg/mL) on NIH3T3 cell were detected by the MTT and Brdu incorporation assay (50, 100, and 150 mg/mL). The effects of EAP (37.5, 75, and 150 mg/mL) on the cell cycle were detected by flow cytometry. The cell signaling pathways of EAP-promoting NIH3T3 cell proliferation were studied by the MTT and Western blot by using different signaling pathway inhibitors. Results: The results showed that EAP (50, 100, and 150 mg/mL) could promote NIH3T3 fibroblasts proliferation (36.4 ± 4.4%, 59.1 ± 4.9%, and 71.5 ± 5.7%). The mechanism of EAP promoting NIH3T3 cell proliferation should be as follows: EAP elevated cyclin D1 expression by activating MEK/ERK signaling pathway, and then promoted cell cycle from G1 to S phase, finally caused the proliferation of NIH3T3 cell. PI3K signaling pathway may be the upstream of MEK/ERK signaling pathway. Discussion and conclusion: The study demonstrates that EAP is effective in promoting effects on proliferation and migration activity of NIH3T3 cell, and the proliferation activity of EAP on NIH3T3 cell may be achieved through the PI3KRacPAKMEK signaling pathway.
ERK, NIH3T3, PI3K, signaling pathway
Introduction Earthworms have been used as a traditional medicine in China, Japan, and other Far East countries from a long time. Modern research found that the whole earthworm has numerous pharmacological effects such as anticoagulation, thrombolytic, anticancer, immunoregulation, antihypertension, antiarrhythmia, antiatheroscloresis, relieving cough and asthma, antibiosis, antioxidation, and promotion of wound healing (Liu & Wang, 2013). In recent years, researches have demonstrated that earthworm extracts could promote wound healing (Grdisa et al., 2001, 2004; Matausijc-Pisl et al., 2010; Zhang et al., 2006). Our earlier study showed that the earthworm protein components are very effective for promoting deep degree burn-wounded SD rats healing without scarring (Zhou et al., 2010). However, there are few articles which involve the mechanism of earthworm protein promoting wound healing.
Received 25 February 2015 Revised 2 July 2015 Accepted 12 July 2015 Published online 1 October 2015
It is believed that fibroblasts are critical in supporting normal wound healing, breaking down the fibrin clot, creating new extra cellular matrix (ECM), and collagen structures to support the other cells associated with effective wound healing, as well as contracting the wound (Darby et al., 2014). In our study, we investigated the effects of earthworm active protein (EAP) on mouse embryonic fibroblast (NIH/3T3) proliferation and its mechanisms. The active protein (EAP) was separated and purified from fresh earthworms followed by exploring the proliferation activity of NIH3T3 cell.
Materials and methods Materials The materials are mouse embryos fibroblasts cell line (NIH3T3) (China Center for Type Culture Collection at
*These authors are joint first authors. Marine College Shandong University (weihai), Weihai, Shandong 264209, China. Correspondence: Aiguo Ji, ! 2015 Taylor & Francis
HISTORY
[email protected]
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S. SONG ET AL.
Wuhan) and earthworm activity protein (EAP): prepared by the International Biotechnology R&D Center of Shandong University at Weihai (Weihai, China).
Downloaded by [University of Otago] at 18:41 22 November 2015
Reagents Dulbecco’s modified Eagle’s medium (DMEM); fetal bovine serum (FBS) (Gibco, Grand Island, NY); 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Shanghai Biological Engineering Company, Shanghai, China); ready-to-use normal goat serum (Boster Company, Wuhan, China); trypsin (Invitrogen, Waltham, MA); RNA enzyme, crystal violet dye, PD98095, U0126, LY294002, RIPA lysissolution BCA kit, Tween20 (Biyuntian, Lijiang, China); bromoceoxyuridine (Brdu) (Sigma, St. Louis, MO); triton-X100 (Aplichem, Darmstadt, Germany); the mice anti-BrdU antibodies (LifeSpan Biosciences, Seattle, WA); DAPI (Aplichem, Darmstadt, Germany); PI, PLX-4720, ZM336372 (Selleck); PMSF (Amresco, Solon, OH); protein color marker (Fermentas, Burlington, Canada); p-ERKantibody, ERKantibody, p-Akt antibody, Aktantibody (Cell Signaling Technology, Danvers, MA); Cyclin D1 antibody, rapid StepTM ECL antibody (Millipore, Billerica, MA); GAPDH antibody; the goat anti-rabbit antibodyIgG (H+L); HRP. EAP’s impact on proliferation and migration of NIH3T3 cell
hole, growing under the condition of 37 ± 0.5 C and 5% CO2. BrdU was added in dark, with the final concentration of 10 mM, discarding the culture medium after 64 h and operating a series of strategies according to the method described by the reagent kit. After observation, pictures were taken randomly in different views under a fluorescent inverted microscope. Finally, synthesis of fluorescent (Figure 2) was observed using the NIH Image J software and the percentage of BrdU-positive cells taking in total cell number was calculated (Guo et al., 2012; Kee et al., 2002). Determination of the effects on the cell cycle Pretreatment of cells was done according to the methods described above. The final concentrations of drugs were 0, 37.5, 75, and 150 mg/mL. Cells were collected after being exposed to drug for 64 h, centrifuged (1000 rpm, 5 min) and washed with PBS, again centrifuged twice to discharge the supernatant, and washed with 150 mL PBS. 350 mL of 100% ethanol was slowly dripped to 70%, at 4 C for 12 h. Then, PBS was added and centrifuged (1000 rpm, 5 min), and washed two times at least. The supernatant was discharged and 10 mg/mL RNase of 1.5 mL was added with a final concentration of 150 mg/mL and incubated in a cell culture at 37 ± 0.5 C for 30–45 min after sealing in aluminum foil. After adding 1 mL PI (10 mg/mL) in each tube, it was stored at 4 C for 1 h, and 300 mL PBS was added.
Cell viability assay
The change of the cell-cycle protein expression
Cells were seeded at a density of 3000–5000 cell/well in 96-well plates. Cell viability was determined by the MTT reduction assay. After various (0, 25, 50, 100, 150, 200, 250, 300, 350, and 400 mg/mL) indicated treatments for 24 h, the medium was removed and cells were incubated with MTT (5 mg/mL) for 4 h at 37 ± 0.5 C. The dark blue formazan crystals that formed in intact cells were solubilized with DMSO, and then the absorbance was measured at 570 nm on a microplate reader.
All the proteins in cells were extracted and the expression of target protein, cyclin D1, was detected with Western blotting.
Proliferation activity assay The cell was inoculated in a 24-hole culture plate under an appropriate density; ordinary culture medium was replaced with a basic culture medium to incubate for 24 h before stimulating the cell with drugs, and then basic culture medium was abandoned. Accurate concentration of drugs dissolved in medium were added one by one according to the experimental group, 100 mL per
The study of cell signaling pathways The effect of MEK inhibitors on EAP’s promoting function on NIH3T3 cell proliferation Cells were seeded at a density of 3000–5000 cell/well in 96-well plates. They were pretreat according to the methods described above. The experiment was divided into six groups: blank control group, 150 mg/mL EAP group, PD98059 (MEK1 inhibitor) group, U0126 (MEK1/2 inhibitor) group, EAP+PD98059 group, and EAP+U0126 group. All the experimental groups were incubated by its corresponding inhibitor for 1 h before being exposed to 150 mg/mL EAP, 37 ± 0.5 C, 5% CO2, 68 h and the cell survival rate was detected with MTT (Chen et al., 2014).
EARTHWORM ACTIVE PROTEIN ON FIBROBLAST PROLIFERATION
The effect of MEK inhibitors on expression of the key protein of NIH3T3 cell proliferation with Western blotting The expression of p-ERK protein in the NIH3T3 cell was detected after incubating with MEK inhibitors. In addition to this, the total protein from the cell was extracted into a basic culture medium incubated with blank, inhibitor, best concentration of EAP, and inhibitor combined with EAP for 68 h. The expression of target protein p-ERK was detected.
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Detect the inhibitor Raf’s impact on EAP’s promoting function on cell proliferation According to the signal pathway (Kolch, 2000), priority is given to B-Raf and RAF-1 inhibitors because they are located in the upstream position in the same way. The experiment was divided into six parallel groups: blank control group, 150 mg/mL EAP group, PLX-4720 (B-Raf inhibitor) group, ZM336372 (Raf-1 inhibitor) group, EAP + PLX-4720 group, and EAP + ZM336372 group. All the experimental groups were pretreated with the corresponding inhibitors for 1 h, followed by 150 mg/mL EAP, 37 ± 0.5 C, 5% CO2, and 68 h, and the cell survival rate was detected with MTT. PI3K inhibitor’s impact on EAP’s promoting function on NIH3T3 cell proliferation From the procedure mentioned above, MEK signaling pathway may be activated by PI3KRacPAKMEK (Chang et al., 2011; Fu et al., 2009). The effect of PI3K inhibitor on the cell survival rate was detected with MTT. The experiment was divided into six parallel groups: blank control group, 150 mg/mL EAP group, LY294002 (PI3K inhibitor) group, and EAP + LY294002 group. All the experimental groups were pretreated with the LY294002 for 1 h, followed by 150 mg/mL EAP, 37 ± 0.5 C, 5% CO2, and 68 h; and the cell survival rate was detected with MTT.
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followed by post hoc tests. Statistical tests were performed using the SPSS 13.0 software (SPSS Inc., Chicago, IL). A p value of 50.05 was considered to be statistically significant.
Results The earthworm active protein’s effects on cell proliferation and migration To determine the best EAP concentration promoting NIH3T3 cell proliferation The experimental results show that the cell proliferation rate after 68 h incubation under the condition of a certain drug concentration between 0 and 200 mg/mL increases with the drug dose. The increase of cell proliferation rate is significance when the concentration is greater than 50 mg/mL and the proliferation rate reaches the peak between the concentrations of 150–200 mg/mL. From what has been discussed above, we prefer 150 mg/mL EAP as the best drug concentration (Figure 1). Effects of EAP on replication of DNA in the NIH3T3 cell examined by BrdU incorporation The results of BrdU staining suggest that only a few cells exhibit positive results in the control group; however, the percentage of the BrdU+ cells in the groups treated with EAP (50, 100, and 150 mg/mL) were 36.4 ± 4.4%, 59.1 ± 4.9%, and 71.5 ± 5.7%; these values are significantly higher than the control group (Figure 2). Effects of EAP on the NIH3T3 cell cycle The results of the flow cytometric assay showed that EAP can increase the proportion of cells in S phase in a dosedependent manner after the cells were cultured for
PI3K signaling pathway inhibitors’ impact on the expression of key protein related to cell proliferation Protein pERK and pAKT expression levels in NIH3T3 cell before and after incubation with inhibitors were detected. Statistical analysis Results are expressed as the mean ± SD. Differences between groups were assessed by one-way ANOVA
Figure 1. Different concentrations of EAP effects on cell proliferation rate. Data are presented as mean ± SEM of three independent experiments. **p50.01 versus control.
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Figure 2. Change in the number of BrdU+ NIH3T3 cell treated with different concentrations of EAP. (A, B, C, D) NIH3T3 cell were labeled by BrdU after treated with maintenance medium containing 0, 50, 100, and 150 mg/mL EAP for 48 h, respectively. The proliferating cells show BrdU positive (green) and DAPi labeled the cell nucleus (blue). (E) Statistical analysis of BrdU+ cells of different groups. The ratio between the BrdU+ cells and DAPi-staining cell nucleus was viewed as the evaluation criterion. The data were represented as mean ± standard deviation (SD). **p 50.01 versus control.
68 h (Figure 3). The above results suggest that EAP can dose dependently promote NIH3T3 cell from G1 phase into the S phase and accelerate the process of the cell proliferation (Figure 3). Effects of EAP on expression of cyclins in NIH3T3 cell Cyclins are positive regulatory factors of the cyclindependent kinases, CDKs. The expression of cyclin D1 can increase in the early of G1 phase, combine with
CDK4 and CDK6, phosphorylate downstream proteins, activate DNA synthesis, and then promote cells from G1 phase into S phase. After treatment with EAP, the expression level of cyclin D1 was examined with western blotting (Figure 4A). The results suggest that the expression of cyclin D1 in NIH3T3 cells increased in a dose-dependent manner after being treated with EAP. The expression of cyclin D1 treated with 150 mg/mL EAP has been improved significantly compared with the control group.
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EARTHWORM ACTIVE PROTEIN ON FIBROBLAST PROLIFERATION
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Figure 3. The cell cycle distribution of NIH3T3 cell after treated with different concentrations of EAP. (A) Control group; (B–D) experimental groups treated with 50, 100, and 150 mg/mL EAP, respectively.
Specific signaling pathway of EAP’s proliferation activity and its effect on cell cycle Effect of MEK inhibitor on EAP’s proliferation activity in NIH3T3 cell by MTT assay Adding the MEK inhibitor PD98059 and U0126 separately was proved to inhibit the EAP’s cell proliferation activity in NIH3T3 cells (Figure 5A) without showing any cell toxicity or changing the cell morphology obviously. Thus, we infer that MEK/ERK signaling pathway is associated with the EAP’s proliferation activity in NIH3T3 cell. Effect of MEK inhibitor on the expression of key protein involved in the NIH3T3 cell proliferation Western blotting showed that EAP significantly improved the phosphorylated level of p-ERK, whereas
PD98059 and U0126 blocked the phosphorylation of ERK (Figure 4C and D). In conclusion, our results indicate that the ERK signaling pathway is associated with EAP’s proliferation activity in NIH3T3 cell. Effect of Raf inhibitor on EAP’s proliferation activity in NIH3T3 cell Above research results demonstrate that EAP promotes NIH3T3 cell proliferation through MEK/ERK signaling pathway. According to the classical Raf/MEK/ERK signaling pathway, we infer that B-Raf and/or Raf-1 may participate in the activation progress. Later on MTT assay was performed to examine how Raf inhibitor works on EAP’s proliferation activity in NIH3T3 cells. Our data show that PLX-4720 (B-Raf inhibitor) or ZM336372 (Raf-1 inhibitor) did not block EAP’s proliferation activity evidently (Figure 5B).
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Figure 4. Results of Western blot. (A) Expression of cyclin D1 in NIH3T3 cell after treated with EAP. (B) pERK and pAkt expression changes in NIH3T3 cell after treated with LY294002. (C) Effect of MEK1 inhibitor PD98059 on the expression of p-ERK protein in NIH3T3 cell. (D) Effect of MEK1/2 inhibitor U0126 on the expression of p-ERK protein in NIH3T3 cell. Data are presented as mean ± SD of three independent experiments. **p 5 0.01.
Effect of PI3K inhibitor on EAP’s proliferation activity in NIH3T3 cells The MTT assay was performed to examine how PI3K inhibitor LY29400 works on EAP’s proliferation activity in NIH3T3 cells. The results show that LY29400 strongly inhibited EAP’s proliferation activity (Figure 5C).
Western blot analysis was used to detect the expression of pERK and pAkt. It was observed that after treatment with EAP, their expression increased significantly compared with the control group (p50.01). This effect of EAP was obviously inhibited by LY29400 (p50.01) (Figure 4B). Since previous results (Figure 5B) have demonstrated the disability of B-Raf and Raf-1 to block
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EARTHWORM ACTIVE PROTEIN ON FIBROBLAST PROLIFERATION
Figure 5. Effect of inhibitors on EAP’s proliferation activity in NIH3T3 cell by MTT assay. Data are presented as mean ± SD of three independent experiments. **p 5 0.01 versus EAP.
the cell proliferation of NIH3T3, we infer that the proliferation progress was probably conducted through the PI3KRacPAKMEK signaling pathway (Figures 4B and 5C).
Discussion Wound healing is an intricate process where the skin or other body tissue repairs itself after injury. This process is divided into predictable phases: blood clotting,
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inflammation, the growth of new tissue (proliferation), and the remodeling of tissue. Fibroblasts play an important role in the process of wound healing, formation of granulation tissue, and vascular regeneration processes. Additionally, fibroblasts can proliferate, migrate, synthesize, and secrete large quantities of collagen and extracellular matrix, participate in the formation of granulation tissue, which is the key contribution for wound healing. In this manuscript, our research demonstrates that EAP can promote NIH3T3 cell transfer from G1-phase into S-phase, increase the proportion of cells in S-phase, and upregulate the DNA replication in a dose-dependent manner. cyclin D1 expression in NIH3T3 cell was also proved to increase after treatment with EAP. These results efficiently explained EAP’s promoting the function of proliferation activity. It is believed that cell proliferation process is regulated by multiple signaling pathways. One study showed that PI3K participate in the Schwann cells proliferation process promoted by the earthworm extracts. Li-Juan Fu illustrated that Gd could promote NIH3T3 cell get into S phase through ERK and PI3K signaling pathway. Molecular mechanism of EAP’s promoting effect on cells proliferation activity of NIH3T3 cell remains unknown. This study first explored part of these molecular mechanisms that EAP can up-regulate p-ERK level and this effect can be inhibited by U0126. Also, this result demonstrates that EAP’s proliferation activity is related to the ERK signaling pathway. Similarly, EAP can upregulate p-Akt level and this effect can be inhibited by LY294002. Additionally, this result demonstrates that EAP’s proliferation activity is related to the PI3K signaling pathway. Thus, we believe that EAP can activate MEK/ERK signaling pathway, conduct cyclin D1 expression, promote cells transfer from G1 phase to S phase, and consequently accelerate the proliferation. Above all, the proliferation activity of EAP on NIH3T3 cell may be achieved through the PI3KRacPAKMEK signaling pathway. In addition, this paper also demonstrates EAP’s promoting effect on proliferation and migration activity of NIH3T3 cell and provides theoretical basis for its role in the process of wound healing. Our results showed the proliferation progress can be affected by cyclin D1, but cannot rule out the possibility of other cytokines’ participation. We only proved that the PI3K signaling pathway could promote cell proliferation by affecting phosphorylation of ERK. The specific action between PI3K and p-ERK remains unclear, which needs to be studied on the cell surface the receptor EAP acts on.
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Declaration of interest This project was supported by the National Natural Science Foundation of China (Grant no. 81371455).
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References Chang YM, Kuo WH, Lai TY, et al. (2011). RSC96 Schwann cell proliferation and survival induced by dilong through PI3K/Akt signaling mediated by IGF-I. Evid Based Complement Alternat Med 2011:216148. Chen Y, Li T, Yu X, et al. (2014). The RTK/ERK pathway is associated with prostate cancer risk on the SNP level: A pooled analysis of 41 sets of data from case-control studies. Gene 534:286–97. Darby IA, Laverdet B, Bonte F, et al. (2014). Fibroblasts and myofibroblasts in wound healing. Clin Cosmet Investig Dermatol 7:301–11. Fu LJ, Li JX, Yang XG, et al. (2009). Gadolinium-promoted cell cycle progression with enhanced S-phase entry via activation of both ERK and PI3K signaling pathways in NIH 3T3 cells. J Biol Inorg Chem 14:219–27. Grdisa M, Popovic M, Hrzenjak T. (2001). Glycolipoprotein extract (G-90) from earthworm Eisenia foetida exerts some antioxidative activity. Comp Biochem Physiol A Mol Integr Physiol 128:821–5.
Grdisa M, Popovic M, Hrzenjak T. (2004). Stimulation of growth factor synthesis in skin wounds using tissue extract (G-90) from the earthworm Eisenia foetida. Cell Biochem Funct 22:373–8. Guo JJ, Shan LD, Wu G. (2012). Improvement in technique of BrdU immunohistochemical staining by bleach section. China Med Herald 9:16–20. Kee N, Sivalingam S, Boonstra R, et al. (2002). The utility of Ki-67 and BrdU as proliferative markers of adult neurogenesis. J Neurosci Methods 115:97–105. Kolch W. (2000). Meaningful relationships: The regulation of the Ras/Raf/MEK/ERK pathway by protein interactions. Biochem J 351:289–305. Liu WY, Wang SD. (2013). Advance in the pharmacological effects of earthworm. Zhongguo Zhong Xi Yi Jie He Za Zhi 33:282–5. Matausijc-Pisl M, Cupic H, Kasuba V, et al. (2010). Tissue extract from Eisenia foetida as a wound-healing agent. Eur Rev Med Pharmacol Sci 14:177–84. Zhang M, Li X, Liu Y, et al. (2006). Effects of extract of dilong (pheretima) on the scalded skin in rats. J Tradit Chin Med 26:68–71. Zhou Y, Xi W, Song SL, et al. (2010). EAP’s promoting healing activity on deep second degree scald wounds in rats. Chin J Trauma 26:377–9.
ISSN: 1388-0209 (Print) 1744-5116 (Online) Journal homepage: http://www.tandfonline.com/loi/iphb20
Effect of earthworm active protein on fibroblast proliferation and its mechanism Shuliang Song, Yuling Wang, Kai Ji, Hao Liang & Aiguo Ji To cite this article: Shuliang Song, Yuling Wang, Kai Ji, Hao Liang & Aiguo Ji (2015): Effect of earthworm active protein on fibroblast proliferation and its mechanism, Pharmaceutical Biology, DOI: 10.3109/13880209.2015.1073333 To link to this article: http://dx.doi.org/10.3109/13880209.2015.1073333
Published online: 07 Oct 2015.
Submit your article to this journal
Article views: 6
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=iphb20 Download by: [University of Otago]
Date: 22 November 2015, At: 18:41
PHARMACEUTICAL BIOLOGY 2015; EARLY ONLINE: 1–8 http://dx.doi.org/10.3109/13880209.2015.1073333
SHORT COMMUNICATION
Effect of earthworm active protein on fibroblast proliferation and its mechanism Shuliang Song1*, Yuling Wang1*, Kai Ji2, Hao Liang1, and Aiguo Ji1,3
Downloaded by [University of Otago] at 18:41 22 November 2015
1 Marine College Shandong University (weihai), Shandong, China, 2Plastic Surgery Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing, China, and 3School of Pharmaceutical Sciences Shandong University, Shandong, China
ABSTRACT
KEYWORDS
Context: Earthworms have been used as a traditional medicine in China from thousands of years. In recent years, research has demonstrated that earthworm extracts might promote wound healing; however, its mechanism is still unknown. Objective: The study investigates the mechanism and effects of earthworm active protein (EAP), on mouse embryonic fibroblast (NIH/3T3) proliferation. Materials and methods: The effects of earthworm active protein (EAP) in different concentrations (0, 25, 50, 100, 150, and 200 mg/mL) on NIH3T3 cell were detected by the MTT and Brdu incorporation assay (50, 100, and 150 mg/mL). The effects of EAP (37.5, 75, and 150 mg/mL) on the cell cycle were detected by flow cytometry. The cell signaling pathways of EAP-promoting NIH3T3 cell proliferation were studied by the MTT and Western blot by using different signaling pathway inhibitors. Results: The results showed that EAP (50, 100, and 150 mg/mL) could promote NIH3T3 fibroblasts proliferation (36.4 ± 4.4%, 59.1 ± 4.9%, and 71.5 ± 5.7%). The mechanism of EAP promoting NIH3T3 cell proliferation should be as follows: EAP elevated cyclin D1 expression by activating MEK/ERK signaling pathway, and then promoted cell cycle from G1 to S phase, finally caused the proliferation of NIH3T3 cell. PI3K signaling pathway may be the upstream of MEK/ERK signaling pathway. Discussion and conclusion: The study demonstrates that EAP is effective in promoting effects on proliferation and migration activity of NIH3T3 cell, and the proliferation activity of EAP on NIH3T3 cell may be achieved through the PI3KRacPAKMEK signaling pathway.
ERK, NIH3T3, PI3K, signaling pathway
Introduction Earthworms have been used as a traditional medicine in China, Japan, and other Far East countries from a long time. Modern research found that the whole earthworm has numerous pharmacological effects such as anticoagulation, thrombolytic, anticancer, immunoregulation, antihypertension, antiarrhythmia, antiatheroscloresis, relieving cough and asthma, antibiosis, antioxidation, and promotion of wound healing (Liu & Wang, 2013). In recent years, researches have demonstrated that earthworm extracts could promote wound healing (Grdisa et al., 2001, 2004; Matausijc-Pisl et al., 2010; Zhang et al., 2006). Our earlier study showed that the earthworm protein components are very effective for promoting deep degree burn-wounded SD rats healing without scarring (Zhou et al., 2010). However, there are few articles which involve the mechanism of earthworm protein promoting wound healing.
Received 25 February 2015 Revised 2 July 2015 Accepted 12 July 2015 Published online 1 October 2015
It is believed that fibroblasts are critical in supporting normal wound healing, breaking down the fibrin clot, creating new extra cellular matrix (ECM), and collagen structures to support the other cells associated with effective wound healing, as well as contracting the wound (Darby et al., 2014). In our study, we investigated the effects of earthworm active protein (EAP) on mouse embryonic fibroblast (NIH/3T3) proliferation and its mechanisms. The active protein (EAP) was separated and purified from fresh earthworms followed by exploring the proliferation activity of NIH3T3 cell.
Materials and methods Materials The materials are mouse embryos fibroblasts cell line (NIH3T3) (China Center for Type Culture Collection at
*These authors are joint first authors. Marine College Shandong University (weihai), Weihai, Shandong 264209, China. Correspondence: Aiguo Ji, ! 2015 Taylor & Francis
HISTORY
[email protected]
2
S. SONG ET AL.
Wuhan) and earthworm activity protein (EAP): prepared by the International Biotechnology R&D Center of Shandong University at Weihai (Weihai, China).
Downloaded by [University of Otago] at 18:41 22 November 2015
Reagents Dulbecco’s modified Eagle’s medium (DMEM); fetal bovine serum (FBS) (Gibco, Grand Island, NY); 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Shanghai Biological Engineering Company, Shanghai, China); ready-to-use normal goat serum (Boster Company, Wuhan, China); trypsin (Invitrogen, Waltham, MA); RNA enzyme, crystal violet dye, PD98095, U0126, LY294002, RIPA lysissolution BCA kit, Tween20 (Biyuntian, Lijiang, China); bromoceoxyuridine (Brdu) (Sigma, St. Louis, MO); triton-X100 (Aplichem, Darmstadt, Germany); the mice anti-BrdU antibodies (LifeSpan Biosciences, Seattle, WA); DAPI (Aplichem, Darmstadt, Germany); PI, PLX-4720, ZM336372 (Selleck); PMSF (Amresco, Solon, OH); protein color marker (Fermentas, Burlington, Canada); p-ERKantibody, ERKantibody, p-Akt antibody, Aktantibody (Cell Signaling Technology, Danvers, MA); Cyclin D1 antibody, rapid StepTM ECL antibody (Millipore, Billerica, MA); GAPDH antibody; the goat anti-rabbit antibodyIgG (H+L); HRP. EAP’s impact on proliferation and migration of NIH3T3 cell
hole, growing under the condition of 37 ± 0.5 C and 5% CO2. BrdU was added in dark, with the final concentration of 10 mM, discarding the culture medium after 64 h and operating a series of strategies according to the method described by the reagent kit. After observation, pictures were taken randomly in different views under a fluorescent inverted microscope. Finally, synthesis of fluorescent (Figure 2) was observed using the NIH Image J software and the percentage of BrdU-positive cells taking in total cell number was calculated (Guo et al., 2012; Kee et al., 2002). Determination of the effects on the cell cycle Pretreatment of cells was done according to the methods described above. The final concentrations of drugs were 0, 37.5, 75, and 150 mg/mL. Cells were collected after being exposed to drug for 64 h, centrifuged (1000 rpm, 5 min) and washed with PBS, again centrifuged twice to discharge the supernatant, and washed with 150 mL PBS. 350 mL of 100% ethanol was slowly dripped to 70%, at 4 C for 12 h. Then, PBS was added and centrifuged (1000 rpm, 5 min), and washed two times at least. The supernatant was discharged and 10 mg/mL RNase of 1.5 mL was added with a final concentration of 150 mg/mL and incubated in a cell culture at 37 ± 0.5 C for 30–45 min after sealing in aluminum foil. After adding 1 mL PI (10 mg/mL) in each tube, it was stored at 4 C for 1 h, and 300 mL PBS was added.
Cell viability assay
The change of the cell-cycle protein expression
Cells were seeded at a density of 3000–5000 cell/well in 96-well plates. Cell viability was determined by the MTT reduction assay. After various (0, 25, 50, 100, 150, 200, 250, 300, 350, and 400 mg/mL) indicated treatments for 24 h, the medium was removed and cells were incubated with MTT (5 mg/mL) for 4 h at 37 ± 0.5 C. The dark blue formazan crystals that formed in intact cells were solubilized with DMSO, and then the absorbance was measured at 570 nm on a microplate reader.
All the proteins in cells were extracted and the expression of target protein, cyclin D1, was detected with Western blotting.
Proliferation activity assay The cell was inoculated in a 24-hole culture plate under an appropriate density; ordinary culture medium was replaced with a basic culture medium to incubate for 24 h before stimulating the cell with drugs, and then basic culture medium was abandoned. Accurate concentration of drugs dissolved in medium were added one by one according to the experimental group, 100 mL per
The study of cell signaling pathways The effect of MEK inhibitors on EAP’s promoting function on NIH3T3 cell proliferation Cells were seeded at a density of 3000–5000 cell/well in 96-well plates. They were pretreat according to the methods described above. The experiment was divided into six groups: blank control group, 150 mg/mL EAP group, PD98059 (MEK1 inhibitor) group, U0126 (MEK1/2 inhibitor) group, EAP+PD98059 group, and EAP+U0126 group. All the experimental groups were incubated by its corresponding inhibitor for 1 h before being exposed to 150 mg/mL EAP, 37 ± 0.5 C, 5% CO2, 68 h and the cell survival rate was detected with MTT (Chen et al., 2014).
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The effect of MEK inhibitors on expression of the key protein of NIH3T3 cell proliferation with Western blotting The expression of p-ERK protein in the NIH3T3 cell was detected after incubating with MEK inhibitors. In addition to this, the total protein from the cell was extracted into a basic culture medium incubated with blank, inhibitor, best concentration of EAP, and inhibitor combined with EAP for 68 h. The expression of target protein p-ERK was detected.
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Detect the inhibitor Raf’s impact on EAP’s promoting function on cell proliferation According to the signal pathway (Kolch, 2000), priority is given to B-Raf and RAF-1 inhibitors because they are located in the upstream position in the same way. The experiment was divided into six parallel groups: blank control group, 150 mg/mL EAP group, PLX-4720 (B-Raf inhibitor) group, ZM336372 (Raf-1 inhibitor) group, EAP + PLX-4720 group, and EAP + ZM336372 group. All the experimental groups were pretreated with the corresponding inhibitors for 1 h, followed by 150 mg/mL EAP, 37 ± 0.5 C, 5% CO2, and 68 h, and the cell survival rate was detected with MTT. PI3K inhibitor’s impact on EAP’s promoting function on NIH3T3 cell proliferation From the procedure mentioned above, MEK signaling pathway may be activated by PI3KRacPAKMEK (Chang et al., 2011; Fu et al., 2009). The effect of PI3K inhibitor on the cell survival rate was detected with MTT. The experiment was divided into six parallel groups: blank control group, 150 mg/mL EAP group, LY294002 (PI3K inhibitor) group, and EAP + LY294002 group. All the experimental groups were pretreated with the LY294002 for 1 h, followed by 150 mg/mL EAP, 37 ± 0.5 C, 5% CO2, and 68 h; and the cell survival rate was detected with MTT.
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followed by post hoc tests. Statistical tests were performed using the SPSS 13.0 software (SPSS Inc., Chicago, IL). A p value of 50.05 was considered to be statistically significant.
Results The earthworm active protein’s effects on cell proliferation and migration To determine the best EAP concentration promoting NIH3T3 cell proliferation The experimental results show that the cell proliferation rate after 68 h incubation under the condition of a certain drug concentration between 0 and 200 mg/mL increases with the drug dose. The increase of cell proliferation rate is significance when the concentration is greater than 50 mg/mL and the proliferation rate reaches the peak between the concentrations of 150–200 mg/mL. From what has been discussed above, we prefer 150 mg/mL EAP as the best drug concentration (Figure 1). Effects of EAP on replication of DNA in the NIH3T3 cell examined by BrdU incorporation The results of BrdU staining suggest that only a few cells exhibit positive results in the control group; however, the percentage of the BrdU+ cells in the groups treated with EAP (50, 100, and 150 mg/mL) were 36.4 ± 4.4%, 59.1 ± 4.9%, and 71.5 ± 5.7%; these values are significantly higher than the control group (Figure 2). Effects of EAP on the NIH3T3 cell cycle The results of the flow cytometric assay showed that EAP can increase the proportion of cells in S phase in a dosedependent manner after the cells were cultured for
PI3K signaling pathway inhibitors’ impact on the expression of key protein related to cell proliferation Protein pERK and pAKT expression levels in NIH3T3 cell before and after incubation with inhibitors were detected. Statistical analysis Results are expressed as the mean ± SD. Differences between groups were assessed by one-way ANOVA
Figure 1. Different concentrations of EAP effects on cell proliferation rate. Data are presented as mean ± SEM of three independent experiments. **p50.01 versus control.
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Figure 2. Change in the number of BrdU+ NIH3T3 cell treated with different concentrations of EAP. (A, B, C, D) NIH3T3 cell were labeled by BrdU after treated with maintenance medium containing 0, 50, 100, and 150 mg/mL EAP for 48 h, respectively. The proliferating cells show BrdU positive (green) and DAPi labeled the cell nucleus (blue). (E) Statistical analysis of BrdU+ cells of different groups. The ratio between the BrdU+ cells and DAPi-staining cell nucleus was viewed as the evaluation criterion. The data were represented as mean ± standard deviation (SD). **p 50.01 versus control.
68 h (Figure 3). The above results suggest that EAP can dose dependently promote NIH3T3 cell from G1 phase into the S phase and accelerate the process of the cell proliferation (Figure 3). Effects of EAP on expression of cyclins in NIH3T3 cell Cyclins are positive regulatory factors of the cyclindependent kinases, CDKs. The expression of cyclin D1 can increase in the early of G1 phase, combine with
CDK4 and CDK6, phosphorylate downstream proteins, activate DNA synthesis, and then promote cells from G1 phase into S phase. After treatment with EAP, the expression level of cyclin D1 was examined with western blotting (Figure 4A). The results suggest that the expression of cyclin D1 in NIH3T3 cells increased in a dose-dependent manner after being treated with EAP. The expression of cyclin D1 treated with 150 mg/mL EAP has been improved significantly compared with the control group.
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Figure 3. The cell cycle distribution of NIH3T3 cell after treated with different concentrations of EAP. (A) Control group; (B–D) experimental groups treated with 50, 100, and 150 mg/mL EAP, respectively.
Specific signaling pathway of EAP’s proliferation activity and its effect on cell cycle Effect of MEK inhibitor on EAP’s proliferation activity in NIH3T3 cell by MTT assay Adding the MEK inhibitor PD98059 and U0126 separately was proved to inhibit the EAP’s cell proliferation activity in NIH3T3 cells (Figure 5A) without showing any cell toxicity or changing the cell morphology obviously. Thus, we infer that MEK/ERK signaling pathway is associated with the EAP’s proliferation activity in NIH3T3 cell. Effect of MEK inhibitor on the expression of key protein involved in the NIH3T3 cell proliferation Western blotting showed that EAP significantly improved the phosphorylated level of p-ERK, whereas
PD98059 and U0126 blocked the phosphorylation of ERK (Figure 4C and D). In conclusion, our results indicate that the ERK signaling pathway is associated with EAP’s proliferation activity in NIH3T3 cell. Effect of Raf inhibitor on EAP’s proliferation activity in NIH3T3 cell Above research results demonstrate that EAP promotes NIH3T3 cell proliferation through MEK/ERK signaling pathway. According to the classical Raf/MEK/ERK signaling pathway, we infer that B-Raf and/or Raf-1 may participate in the activation progress. Later on MTT assay was performed to examine how Raf inhibitor works on EAP’s proliferation activity in NIH3T3 cells. Our data show that PLX-4720 (B-Raf inhibitor) or ZM336372 (Raf-1 inhibitor) did not block EAP’s proliferation activity evidently (Figure 5B).
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Figure 4. Results of Western blot. (A) Expression of cyclin D1 in NIH3T3 cell after treated with EAP. (B) pERK and pAkt expression changes in NIH3T3 cell after treated with LY294002. (C) Effect of MEK1 inhibitor PD98059 on the expression of p-ERK protein in NIH3T3 cell. (D) Effect of MEK1/2 inhibitor U0126 on the expression of p-ERK protein in NIH3T3 cell. Data are presented as mean ± SD of three independent experiments. **p 5 0.01.
Effect of PI3K inhibitor on EAP’s proliferation activity in NIH3T3 cells The MTT assay was performed to examine how PI3K inhibitor LY29400 works on EAP’s proliferation activity in NIH3T3 cells. The results show that LY29400 strongly inhibited EAP’s proliferation activity (Figure 5C).
Western blot analysis was used to detect the expression of pERK and pAkt. It was observed that after treatment with EAP, their expression increased significantly compared with the control group (p50.01). This effect of EAP was obviously inhibited by LY29400 (p50.01) (Figure 4B). Since previous results (Figure 5B) have demonstrated the disability of B-Raf and Raf-1 to block
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Figure 5. Effect of inhibitors on EAP’s proliferation activity in NIH3T3 cell by MTT assay. Data are presented as mean ± SD of three independent experiments. **p 5 0.01 versus EAP.
the cell proliferation of NIH3T3, we infer that the proliferation progress was probably conducted through the PI3KRacPAKMEK signaling pathway (Figures 4B and 5C).
Discussion Wound healing is an intricate process where the skin or other body tissue repairs itself after injury. This process is divided into predictable phases: blood clotting,
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inflammation, the growth of new tissue (proliferation), and the remodeling of tissue. Fibroblasts play an important role in the process of wound healing, formation of granulation tissue, and vascular regeneration processes. Additionally, fibroblasts can proliferate, migrate, synthesize, and secrete large quantities of collagen and extracellular matrix, participate in the formation of granulation tissue, which is the key contribution for wound healing. In this manuscript, our research demonstrates that EAP can promote NIH3T3 cell transfer from G1-phase into S-phase, increase the proportion of cells in S-phase, and upregulate the DNA replication in a dose-dependent manner. cyclin D1 expression in NIH3T3 cell was also proved to increase after treatment with EAP. These results efficiently explained EAP’s promoting the function of proliferation activity. It is believed that cell proliferation process is regulated by multiple signaling pathways. One study showed that PI3K participate in the Schwann cells proliferation process promoted by the earthworm extracts. Li-Juan Fu illustrated that Gd could promote NIH3T3 cell get into S phase through ERK and PI3K signaling pathway. Molecular mechanism of EAP’s promoting effect on cells proliferation activity of NIH3T3 cell remains unknown. This study first explored part of these molecular mechanisms that EAP can up-regulate p-ERK level and this effect can be inhibited by U0126. Also, this result demonstrates that EAP’s proliferation activity is related to the ERK signaling pathway. Similarly, EAP can upregulate p-Akt level and this effect can be inhibited by LY294002. Additionally, this result demonstrates that EAP’s proliferation activity is related to the PI3K signaling pathway. Thus, we believe that EAP can activate MEK/ERK signaling pathway, conduct cyclin D1 expression, promote cells transfer from G1 phase to S phase, and consequently accelerate the proliferation. Above all, the proliferation activity of EAP on NIH3T3 cell may be achieved through the PI3KRacPAKMEK signaling pathway. In addition, this paper also demonstrates EAP’s promoting effect on proliferation and migration activity of NIH3T3 cell and provides theoretical basis for its role in the process of wound healing. Our results showed the proliferation progress can be affected by cyclin D1, but cannot rule out the possibility of other cytokines’ participation. We only proved that the PI3K signaling pathway could promote cell proliferation by affecting phosphorylation of ERK. The specific action between PI3K and p-ERK remains unclear, which needs to be studied on the cell surface the receptor EAP acts on.
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Declaration of interest This project was supported by the National Natural Science Foundation of China (Grant no. 81371455).
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