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Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway
European Journal of Pharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Molecular and cellular pharmacology
Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway Xiaolei Li a,1, Yu Sun b,1, Hui Chen b, Gengyao Zhu b, Yuan Liang b, Qiang Wang b, Jingcheng Wang b,n, Lianqi Yan b,n a b
Department of Orthopedics, The Second Xiangya Hospital Affiliated with Central South University, Changsha 410011, China Department of Orthopedics, Clinical medical college of Yangzhou University, Subei People’s Hospital of Jiangsu Province, Yangzhou 225001, China
art ic l e i nf o
a b s t r a c t
Article history: Received 10 December 2015 Received in revised form 6 April 2016 Accepted 8 April 2016
Hydroxycamptothecin (HCPT) has been proven to prevent intraarticular scar adhesion, but the mechanism is still unclear. ER stress is known to participate in many diseases, and the IRE-1 signal pathway has been reported in fibrotic diseases. The aim of this study was to illustrate the mechanism of HCPTinduced apoptosis in fibroblasts and the prevention of intraarticular scar adhesion. The effects of HCPT on fibroblasts were determined by CCK-8 assay, Hoechst staining and Western blot. The effect of HCPT on intraarticular scar adhesion was detected by macroscopic evaluation, hydroxyproline content, histological evaluation, fibroblast counting and immunohistochemical analysis. HCPT induced apoptosis of fibroblasts, according to CCK-8 assays, Hoechst staining and Western blot analysis. As the concentration of HCPT increased, the expressions of glucose-regulated protein 78 (GRP78), inositol-requiring kinase1 (IRE-1), C/EBP homologous protein (CHOP) and Bax were all increased, but the expression of Bcl-2 was decreased. Knockdown of IRE-1 alleviated the HCPT-induced apoptosis in our fibroblast model. HCPT could prevent intraarticular scar adhesion, according to the results of macroscopic evaluation, hydroxyproline content, histological evaluation and fibroblast counting in a rabbit model. Immunohistochemical analysis showed that IRE-1 expression increased as the concentration increased. The present study showed that the IRE-1 signal pathway might be involved in HCPT-induced apoptosis of fibroblast and might play a role in preventing intraarticular scar adhesion. & 2016 Elsevier B.V. All rights reserved.
Keywords: Fibroblast apoptosis IRE-1 signal pathway Hydroxycamptothecin Intraarticular scar adhesion
1. Introduction Intraarticular scar adhesion is considered as an important factor after knee surgery in joint department. Stiffness and severe functional impairments, as well as poor clinical outcomes, occur after knee surgery as a result of intraarticular scar adhesion. The etiology of intraarticular scar adhesion is unclear, but fibrous tissue hyperplasia is considered by many authors to have a critical role in the formation of intraarticular scar adhesion. Therefore, various anti-proliferation agents were applied to reduce intraarticular scar adhesion in animal experiments and achieved limited success (Fukui et al., 2001; Hayashi et al., 2004; Sun et al., 2014). n
Corresponding author. E-mail addresses: [email protected] (J. Wang), [email protected] (L. Yan). 1 Xiaolei Li and Yu Sun equally contributed to paper.
Hydroxycamptothecin (HCPT) is a cell cycle-specific DNA topoisomerase I inhibitor agent that shows high activity against the proliferation of many tumor cells (Zhou et al., 2011). HPCT is used as an anti-tumor agent against bladder cancer, gastric cancer, colorectal cancer and has had success in the clinic (Yang et al., 2007; Wang et al., 2007). As an anti-tumor agent, HCPT has been shown to induce the apoptosis of human tendon capsule fibroblasts. Moreover, Yang and Sun have reported that HCPT can minimize epidural scar adhesion by preventing the proliferation of fibroblasts in a rat laminectomy model (Sun et al., 2008; Yang et al., 2011). Both mitochondrial dysfunction and the ER stress-mediated cell death pathway are involved in drug-induced apoptosis in mammalian cells (Yen et al., 2012; Shi et al., 2013). Furthermore, previous studies have shown that caspase-3 and caspase-9 play important roles in HCPT-induced apoptosis in human tendon capsule fibroblasts (Tang et al., 2012). These results showed that endoplasmic reticulum (ER) stress
http://dx.doi.org/10.1016/j.ejphar.2016.04.012 0014-2999/& 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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might also be involved in HPCT-induced apoptosis, and further investigation to investigate the possible mechanism of HCPT-induced apoptosis of human fibroblast is warranted. Recently, many studies have shown that ER stress is closely linked to human disease, such as tissue ischemia, Alzheimer's disease, Parkinson's disease and diabetes mellitus (Rozpedek et al., 2015; Yang and Luo, 2015; Harding et al., 2001; Nishitoh et al., 2002). The ER is a multifunctional signaling organelle that plays a critical role in protein folding, handling of misfolded proteins, posttranslational modification and calcium homeostasis (Boyce et al., 2005; Woehlbier et al., 2011). ER stress is triggered by the accumulation of unfolded proteins and altered calcium homeostasis. In response to ER stress, the unfolded protein response (UPR) restores protein homeostasis by suppressing protein translation, inducing ER-related molecular chaperones to promote refolding of unfolded proteins and removing unfolded proteins by activating ER-associated protein degradation (Xu et al., 2005; Merksamer et al., 2010). Inositol-requiring enzyme 1 (IRE-1) was identified as the only sensor of ER stress in yeast and is essential in both animals and plants (Chen and Brandizzi, 2013). Previous results of rat models showed that IRE-1 played a critical role in fibrosis formation in liver cirrhosis (Jiang et al., 2015). The main purpose of this study was to illustrate the effects of IRE-1 on HCPT-induced apoptosis in human fibroblasts and the prevention of intraarticular scar adhesion in rabbit model.
2. Materials and methods The experimental research was approved by the Ethical Committee and Research Committee of Subei Hospital of Jiangsu Province. The primary fibroblast cell line was obtained from GuangZhou Jenino Biotech Co., Ltd. The fibroblasts were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin G and 100 U/mL streptomycin in an atmosphere of 5% CO2 at 37 °C. Fibroblast cells of passages 3 and 6 were selected in all of the experiments. 2.1. HCPT treatment HCPT was purchased from Lishizhen pharmaceutical Co., Ltd (Hubei, China). Human fibroblasts were cultured in 24-well plates, 96-well plates or 10-cm dishes overnight. After washing with PBS, human fibroblast cells were treated with HCPT at various concentrations and for various durations. The control group was treated with PBS. After all HCPT and PBS treatments, cells were washed with PBS three times and cultured for subsequent experiments. 2.2. Cell viability test Cell counting Kit-8 (CCK-8) was applied to determine cell viability. The human fibroblast cells were cultured in triplicate in 96well plates. The fibroblast cells were treated with 0.05 μg/mL HCPT, 0.1 μg/mL HCPT, 0.5 μg/mL HCPT, 1 μg/mL HCPT and 4 μg/mL HCPT, upon which treated cells were maintained in DMEM for 12 h, 24 h,36 h, 48 h and 60 h. Cell viability was determined according to the manufacturer's instructions. Briefly, the CCK-8 solution (10 mL) was added to each well for 2 h at 37 °C, and cells that were stained with the CCK-8, were considered viable and were reported as a percentage of the control. 2.3. Hoechst 33342 staining Fibroblasts were plated in 6-well plates (2 mL, 1 106 per well)
and incubated to adhere overnight at 37 °C. Increasing concentrations of HCPT were then added to each well, and the cells were incubated for another 8 h at 37 °C. The fibroblast monolayer was then washed three times with PBS and stained with 5 mg/mL Hoechst for 30 min at 37 °C in the dark. Following staining, the fibroblasts were washed three times with PBS. The morphological features of apoptosis were observed by fluorescence microscopy (Olympus BX 51) with a magnification of 400. 2.4. Western blot analysis HCPT-treated fibroblasts were lysed in RIPA buffer (Beyotime, Hangzhou, China) according to the manufacturer's instructions, and the lysates were centrifuged at 12000g for 15 min. Supernatants were collected for western blot analysis, and the protein concentration was determined using a BCA Protein Assay Kit (Beyotime, Hangzhou, China). Equal amounts (60 μg/lane) of total protein were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and transferred onto PVDF membranes. Membranes were blocked with 5% skim milk in TBST for 2 h at room temperature and subsequently incubated with the primary antibodies overnight. Anti-78-kDa glucose-regulated protein (GRP78), anti-CHOP, anti-phosphoIRE-1a (S724) antibodies, anti-Bax and anti-Bcl-2 antibodies were obtained from Abcam, USA. Anti-inositol-requiring enzyme 1a (IRE-1a) was obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). Anti-poly ADPribose polymerase (PARP), anti-β-actin antibody were purchased from Cell Signaling Technology (Cell Signaling Technology, USA). The membranes were next washed three times in TBST and incubated with secondary antibodies (Santa Cruz Biotechnology, USA) (diluted 1:5 000) for 2 h. The membrane was visualized via an enhanced ECL system (Millipore, USA). 2.5. Gene silencing using lentiviral infection Lentiviral vectors containing the target gene, IRE-1, were purchased from Shanghai Genechem Co. Ltd. (Genechem, China). Lentiviral infection was carried out according to the manufacturer's instructions. Fibroblasts were incubated with the lentiviral vector at an MOI of 20 in the presence of 2 mg/mL polybrene (Gibco, USA) overnight. Subsequently, the medium was removed, and fresh complete medium was added. Following transfection for 48 h, the fibroblast cells were cultured with 2 mg/mL puromycin (Sigma, USA) for 96 h. Stably transfected cells were then used in subsequent experiments, such as HCPT treatment and western blot and so on. 2.6. Animals Thirty-six mature male New Zealand rabbits weighing 3.8 kg were used in the present study. The rabbits were randomly divided into three groups (twelve rabbits in each group): HCPT (1.0 mg/mL) group, HCPT (0.5 mg/mL) group and control group. The rabbits were housed for 1 week to adapt to the condition of the laboratory before the experiment. 2.7. Animal model An animal model of intraarticular adhesion was performed according to our previous studies (Fukui et al., 2000; Yan et al., 2010). All animals were anesthetized by intravenous administration of pentobarbital sodium, and the animals were fixed in the supine position. The knee joint was opened, and the both sides of the femoral condyle were exposed after the hair was shaved and the exposed skin was sterilized with iodophor. Approximately 10 mm 10 mm squares of the cortical bone were removed from
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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Fig. 1. Application of HCPT inhibits cell viability and induces apoptosis in human fibroblasts. (A) Cell viability after various concentrations of HCPT was detected by CCK-8 assay. The results showed that HCPT could inhibit cell viability in a dose-dependent manner. (B) Cell viability after various durations for 1 μg/mL HCPT treatment were also detected by CCK-8 assay. The results showed that 1 μg/mL HCPT could inhibit cell viability in a time-dependent manner. (C) Fibroblast cells with condensed and fragmented nuclei were observed after 1 μg/mL HCPT treatment, according to the result of Hoechst staining. (D) Western blot analysis showed that HPCT could increase the expression of cleaved PARP in a time-dependent manner. (E) The histogram in this panel represents the mean 7 S.E.M of three independent experiments. *P o 0.05 and **Po 0.01 versus control group (0 μg/mL).
both sides of the femoral condyle until the cancerous bone underneath was exposed. The articular cartilage was left intact. 2.8. Application of drugs Cotton pads (15 mm 15 mm) soaked with HCPT 1.0 mg/mL and 0.5 mg/mL were applied on the exposed decorticated area for 10 min in the corresponding group. Then, the soaked cotton pad was removed, and the surgical site was irrigated with saline. In control group, the decorticated area was only irrigated with saline. In all groups, the surgical site was closed in layers with non-absorbable silk sutures, and the surgical knee joint was fixed extraarticular with Kirschner wire (K-wire) for 4 weeks. 2.9. Macroscopic evaluation Six postoperative rabbits were randomly selected from each group for macroscopic evaluation at 4 weeks. They were killed by intravenous administration of a lethal dose of pentobarbital sodium. The joint was reopened by former skin incision. The presence and severity of adhesion was assessed according to the following visual scoring system by a surgeon who was blinded to the treatment: 0: no adhesions; 1: weak, mild, filmy adhesions that can be eliminated by minimal manual traction; 2: moderate adhesions that can be eliminated by manual traction; 3: dense fibrous adhesions that must be surgically removed.
2.10. Hydroxyproline content After macroscopic evaluation, the scar tissue obtained from the surgical sites was used for biochemical HPC analysis. The assay of hydroxyproline in scar tissue (approximately 20 mg) was determined as previously described by Woessner (Yan et al., 2010). The samples were lyophilized, ground and hydrolyzed with 6 mol/ L HCl at 130 °C for 12 h. Then, they were neutralized with 2.5 N NaOH as indicated with methyl red, and 1 mLof chloramine T was added to the hydrolyzed samples and hydroxyproline standards of four known concentrations. After incubation for 20 min at room temperature, 1 mL of hydroxyproline developer (β-dimethylaminobenzaldehyde solution) was added to the samples and the standards. The absorbance of the solution was measured at 558 nm using a spectrophotometer, and the hydroxyproline content of the adhesion tissue was expressed as micrograms per milligram (μg/mg). 2.11. Histological evaluation Histological evaluation was carried out postoperatively in each group four weeks later. Six remaining rabbits in each group were killed by intravenous injection with a lethal dose of pentobarbital sodium solution. The scar was removed from the joint and embedded in paraffin. First, six sections from each group were stained with
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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Fig. 2. HCPT induces ER stress and the IRE-1 pathway. (A) Western blot analysis showed that 1 μg/mL HPCT could increase the expression of CHOP and Bax and decrease the expression of Bcl-2 in a dose-dependent manner. HCPT could induce apoptosis of fibroblasts in a dose-dependent manner. (B–C) HCPT affected ER stress and the IRE-1 pathway in a dose- and time-dependent manner. Fibroblast cells were treated with different concentrations of HCPT for 24 h or treated with 1 μg/mL HCPT for 24 h and cultured for various durations. The expression levels of GRP-78, P-IRE-1, IRE-1, CHOP and β-actin (loading control) were analyzed by western blot. The histograms represent the mean 7 S.E.M of three independent experiments. *Po 0.05 versus control group (0 μg/mL).
hematoxylin-eosin (H&E). The following two parameters were evaluated under the light microscope with the different magnifications: the intraarticular scar adhesion ( 200) and fibroblasts per 100 mm 100 mm counting area ( 400). Second, six sections of each group were stained with Masson Trichrome. The optical density was evaluated under a light microscope with the magnification of 200. 2.12. Immunohistochemical analysis Six sections of scar tissues were prepared for immunohistochemical analysis of IRE-1. The sections were deparaffinized and rehydrated through gradient ethanol solutions according to a routine avidin–biotin–immunoperoxidase technique. After blocking with goat serum solution for 10 min, these sections were incubated with rabbit-IRE-1 antibody (1:200 dilution as supplied by the manufacturer Abcam) overnight at 4 °C. The sections were then washed in PBS solution, and biotinylated anti-goat lgG was used as secondary antibody for 15 min. The sections were visualized under a light microscope with a magnification of 400
after using 3,3′-diaminobenzidine for 5 min and counterstaining with hematoxylin for 2 min at room temperature. 2.13. Statistical analysis The statistical analysis was performed using SPSS software (version15.0). The data were expressed as the mean 7 S. E. M. ANOVA was used to calculate the significance in hydroxyproline content and fibroblast counting. P-values o0.05 were considered statistically significant in all analyses. 3. Results 3.1. HCPT inhibited the cell viability and induced apoptosis of fibroblasts Human fibroblasts were treated with HCPT at increasing concentrations for 24 h. A Cell Counting Kit-8 (CCK-8) assay demonstrated that HCPT could induce a dramatic loss in the viability of fibroblasts, and cell viability reached a relatively minimal level at 4 μg/mL HCPT. As a result, fibroblasts were treated with HCPT (1 μg/mL) for various durations. CCK-8 assays showed that the cell viability reduction was concomitant with prolonged stimulation. According to the results of
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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Fig. 3. IRE-1 knockdown attenuated the HCPT-induced ER stress and apoptosis in human fibroblasts. (A–B) Human fibroblasts were transduced with IRE-1-mediated lentivirus. Silenced IRE-1 expression was verified by western blot analysis. (C–D) Cell viability and apoptosis in normal and IRE-1-knockdown human fibroblasts were measured by CCK-8 assay and Hoechst staining with 1 μg/mL HCPT treatment for 24 h. After the IRE-1-knockdown in human fibroblasts, cell viability was increased, and the number of apoptotic nuclei was decreased. The data are presented as the mean 7 S.E.M (*Po 0.05). (E–F) The expression levels of the ER stress markers GRP78 and CHOP and the downstream Bcl-2 and Bax were determined by western blot in IRE-1-knockdown and control human fibroblasts after treatment with 1 μg/mL HCPT for 24 h. β-actin was used as the loading control. The histograms in each panel represent the mean 7 S.E.M of three independent experiments. *P o 0.05 versus control group (0 μg/mL).
Table1 The results of intraarticular scar adhesions were evaluated according to the visual scoring system.
which displayed a time dependence with stimulation (Fig. 1C D and E). These results suggest that HCPT could induce apoptosis of human fibroblasts. 3.2. HCPT induced ER stress and activated the IRE-1 pathway
Group
HCPT(1.0 mg/mL) HCPT(0.5 mg/mL) Control
Grade 0
1
2
3
3 1 0
3 3 0
0 2 0
0 0 6
CCK-8 described above, HCPT is an effective inhibitor of fibroblast viability in vitro, and this inhibitory effect is both dose and time dependent (Fig. 1 A and B). Hoechst staining and western blot analysis revealed that fibroblast cells treated with 1 μg/mL HCPT contained condensed and fragmented nuclei and cleaved PARP,
Western blot analysis was used to determine whether ER stress was involved in HCPT-induced apoptosis of fibroblasts. The expression of GRP78 and CHOP, which are inducers of ER stress, was detected (Fig. 2). Likewise, HCPT could decrease antiapoptotic Bcl-2 expression, whereas HCPT increased apoptotic Bax and CHOP expression in a dose-dependent manner. Moreover, the expression of GRP78 increased starting at 0.05 μg/mL HCPT and reached a maximum of 4 μg/mL. IRE-1 was activated from 0.05 μg/mL HCPT and reached a maximum at 4 μg/mL in a dose-dependent manner (Fig. 2 A and B). After treatment with 1 μg/mL HCPT, the GRP78 expression was markedly increased at 3 h after treatment and was sustained for 24 h. Importantly, fibroblasts treated with 1 μg/mL HCPT markedly induced the activation of IRE-1 in a time-dependent manner (Fig. 2C). These data indicated that HCPT induced ER stress and activated the IRE-1 pathway, which might be involved
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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Fig. 4. Hydroxyproline contents of intraarticular scar tissues in the 1 mg/mL HCPT, 0.5 mg/mL HCPT and saline groups. The hydroxyproline content was expressed as μg/mg. *Po 0.05, compared with the hydroxyproline content in the control group. #Po 0.05, compared with the hydroxyproline content in the 0.5 mg/mL HCPT group.
Fig. 6. The fibroblast number in intraarticular scar tissues in the 1 mg/mL HCPT, 0.5 mg/mL HCPT and saline groups. The fibroblast number was expressed as the number/counting area (100 mm 100 mm). *P o0.05, compared with the fibroblast number in the control group. #Po 0.05, compared with the fibroblast number in the 0.5 mg/mL HCPT group.
in apoptosis signaling of human fibroblasts.
3.5. Hydroxyproline contents analysis
3.3. Suppression of HCPT-induced apoptosis of human fibroblast via knockdown of IRE-1
The hydroxyproline contents of the 1.0 mg/mL and 0.5 mg/mL HPCT-treated groups were significantly lower than those of the control group (P o 0.05). Furthermore, the hydroxyproline contents of the 1.0 mg/mL HPCT-treated group were significantly lower than those of the 0.5 mg/mL HPCT-treated group (Po 0.05). The hydroxyproline contents of intraarticular scar tissue in each group are shown in Fig. 4.
Previous results showed that IRE-1 is involved in the expression of CHOP, Bcl-2 and Bax during HCPT-induced ER stress in fibroblast cells. Therefore, we knocked down the IRE-1 gene from fibroblast cells by lentiviral-mediated shRNAs. After successfully silencing the expression of IRE-1 (Fig. 3A and B), fibroblast cells were incubated with 1 μg/mL HCPT as described above. Silencing of IRE-1 resulted in a prominent increase in cell viability according to CCK-8 assays (Fig. 3D) and a decrease in apoptosis according to Hoechst staining (Fig. 3C). Meanwhile, the expression of the ER stress markers (GRP78 and CHOP) and the downstream Bax were decreased and that of Bcl-2 was increased significantly in the IRE-1 silenced group (Fig. 3E and F).
3.4. Macroscopic evaluation of intraarticular adhesion All rabbits recovered without mortality or any sign of wound infection or cutaneous necrosis during the four weeks post operation. After four weeks, macroscopic assessment showed that a significant reduction in the adhesion score was observed in the 1.0 mg/mL and 0.5 mg/mL HPCT-treated groups relative to the controls. In the 1.0 mg/mL HPCT-treated group, filmy membrane-like scar tissue covered the surgical site, which could be dissected easily without bleeding. In the 0.5 mg/mL HCPT group, moderate scar tissue covered the surgical area, which could be dissected by manual traction. In contrast, the control group presented severe thick fibrous tissue around the surgical site, which was dissected with difficulty and was accompanied by bleeding. The results of intraarticular adhesion grading scores according to the visual scoring system are shown in Table 1.
3.6. Histological evaluation In the control group, severe fibrosis and dense fibers with a large population of mature fibroblasts were observed around the surgical site. In the 0.5 mg/mL HPCTtreated group, the surgical site was covered with moderate fibrous adhesion with dense fibroblasts. In contrast, in the 1.0 mg/mL HPCT-treated group, loose scar tissues with fewer fibroblasts were observed around the surgical site. Representative histological images of intraarticular adhesion in rabbits of each group are shown in Fig. 5. 3.7. Effect of 1.0 mg/mL and 0.5 mg/mL HPCT on fibroblasts The fibroblast number in scar tissue from the 1.0 mg/mL and 0.5 mg/mL HPCT groups were significantly less than those of the control group (P o 0.05). Moreover, the fibroblast number of the 1.0 mg/mL HCPT group was significantly lower than that of the 0.5 mg/mL HCPT group (P o 0.05). The fibroblast number in the intraarticular scar tissue of each treatment group is shown in Fig. 6.
3.8. Effect of 1.0 mg/mL HPCT and 0.5 mg/mL HPCT on collagen density The results of Masson's Trichrome staining were in agreement with those of hematoxylin-eosin staining. In the control group, there was dense collagen in the
Fig. 5. Representative histological images of intraarticular adhesion in rabbits treated with 1 mg/mL HCPT, 0.5 mg/mL HCPT or saline. These sections were stained with HE, and the magnification is 200 . In the control group, severe fibrosis and dense fibers with a large population of mature fibroblasts were observed around the surgical site. In the 0.5 mg/mL HPCT-treated group, the surgical site was covered with moderate fibrous adhesion with dense fibroblasts. In contrast, in the 1.0 mg/mL HPCT-treated group, loose scar tissues with fewer fibroblasts were observed around the surgical site.
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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Fig. 7. Collagen density in intraarticular scar tissue in rabbits treated with 1 mg/mL HCPT, 0.5 mg/mL HCPT or saline. These sections were stained with Masson, and the magnification is 200 . The results of Masson's Trichrome staining were agreement with those of hematoxylin-eosin staining. In the control group, there was dense collagen in the intraarticular scar tissue. There was low-density collagen in the intraarticular scar tissue in the 1.0 mg/mL HCPT-treated group, which was decreased compare with that of the 0.5 mg/mL HCPT-treated group.
Fig. 8. The results of expression of IRE-1 in the intraarticular scar tissue treated with 1 mg/mL HCPT, 0.5 mg/mL HCPT and saline were similar to the results of the western blot analysis. The results of IRE-1expression were expressed as mean OD and showed in the histogram. *P o 0.05, compared with the fibroblast number in the control group. #Po 0.05, compared with the fibroblast number in the 0.5 mg/mL HCPT group. intraarticular scar tissue. The collagen density was low in the intraarticular scar tissue from the 1.0 mg/mL HCPT-treated group, which was decreased compared with that of the 0.5 mg/mL HCPT-treated group. The results of collagen density in intraarticular scar tissue in rabbits of each group are shown in Fig. 7.
than the 0.5 mg/mL HPCT-treated group. The results of IRE-1 expression were expressed as mean OD and showed in the histogram. *P o 0.05, compared with the fibroblast number in the control group. #Po 0.05, compared with the fibroblast number in the 0.5 mg/mL HCPT group.
3.9. Immunohistochemical analysis of 1.0 mg/mL and 0.5 mg/mL HPCT on IRE-1 expression
4. Discussion
As shown in Fig. 8, the expression of IRE-1 in the intraarticular scar tissue treated with 1 mg/mL HCPT, 0.5 mg/mL HCPT and saline were similar according to western blot analysis. The expression of IRE-1 in the 1.0 mg/mL and 0.5 mg/mL HPCT-treated groups was higher than that of the control group. Furthermore, the expression of IRE-1 in the 1.0 mg/mL HPCT-treated group was significantly higher
Hydroxycamptothecin is natural alkaloid that is isolated form Camptotheca acuminate. It is widely used to treat cancers with less clinical toxicity and was proven to be an anti-fibrosis agent by previous studies. Previous studies have shown that HCPT could
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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induce apoptosis in various cancer cell, such as lung adenocarcinoma, bladder cancer and colon cancer. In addition, HCPT has also been proven to induce apoptosis of both human tendon capsule and hamster lung fibroblasts (Ping et al., 2006; Yokoo et al., 2007; Zhao et al., 2011). Our previous study showed that topical application of 1 mg/mL HCPT might be the optimal concentration for preventing intraarticular scar adhesion in rabbit models, but the mechanism of HCPT reduction in intraarticular scar adhesion by preventing proliferation and inducing apoptosis in fibroblasts is still unclear. Further experiments are needed to elucidate the precise mechanism (Liang et al., 2014; Yan et al., 2015). In the present study, we found that 0.5 mg/mL and 1.0 mg/mL HCPT could induce fibroblast apoptosis and reduce intraarticular scar adhesion in a rabbit model. Multiple parameters, including the visual scoring system, hydroxyproline content analysis, HE staining, the number of fibroblasts and Masson staining, were applied to evaluate the effect of HCPT on preventing intraarticular scar adhesion in a rabbit model. It is known that fibroblasts can produce collagen and extracellular matrix components in bone defect areas. Uncontrolled production of those may lead to the formation of scar tissue and result in intraarticular adhesion. Furthermore, previous research showed that hydroxyproline accounts for 12.5% of the amino acid content in collagen fiber so that the hydroxyproline content can reflect the formation of collagen in scar tissue (Edwards and O’Brien, 1980). Thus, collagen observation, hydroxyproline content and fibroblast counting could reflect the efficacy of HCPT in preventing intraarticular scar adhesion in a rabbit model. The results of macroscopic evaluation and HE staining showed that there were weak and moderate scar adhesions around the bone defect area in HCPT-treated groups. The results of hydroxyproline content and fibroblast counting were also all significantly lower in the HCPT-treated groups than those in the control group. The results of Masson staining were similar to those of HE staining and showed that little or weak collagen appeared in the bone defect area in the HCPT-treated group. Moreover, the results of the 1 mg/mL HCPT-treated group were also less than those of the 0.5 mg/mL HCPT-treated group. These results indicated that HCPT could prevent scar adhesion after knee surgery and that 1 mg/mL HCPT could have a better effect. In vitro, CCK-8 assays and Hoechst staining were used to elucidate the effect of HCPT on fibroblast proliferation and apoptosis in this study. CCK-8 assays demonstrated that HCPT could inhibit the cell viability of human fibroblasts in a dose-dependent manner and reached minimal cell viability at 4 μg/mL. Moreover, we also found that HCPT could inhibit cell viability in a time-dependent manner, according to CCK-8 assays. Hoechst staining showed that human fibroblasts exhibited condensed and fragmented nuclei when stimulated for 24 h by 1 μg/mL HCPT. The ER is an intracellular organelle and is considered an important site of secretor and membrane protein synthesis, modification and folding into their correct conformations. Furthermore, the ER is responsible for intracellular calcium homeostasis, and ER stress is triggered by imbalances in calcium levels. Cells activate an adaptive mechanism known as the UPR to respond to ER stress. If a cell fails to recover from ER stress, the adaptive response is repressed and apoptosis is triggered by the UPR. The UPR is mediated by three signaling pathways: PKR-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE-1) and activating transcription factor 6 (ATF6) (Shore et al., 2011; Hetz et al., 2012). Previous studies showed that IRE-1 was involved in many different fibrosis-related diseases, and it regulates the proliferation, apoptosis and metabolism of fibroblasts (Mo et al., 2015; Zhao et al., 2014). IRE-1 is one of three ER stress signals and is considered to be a regulator of CHOP transcription, which is a central mediator of ER-stress-induced apoptosis (Chan et al., 2015; Choi
et al., 2015). With the activation of ER stress and accumulation of unfolded proteins, IRE-1 dissociates from GRP/BiP and is permitted to dimerize, which induces splicing of XBP1. The generated spliced XBP1 is a potent transcriptional activator that can induce downstream genes, including those encoding ER chaperones and ERassociated proteins (Lenna et al., 2012; Shi et al., 2013; Coelho et al., 2014). Therefore, western blot analysis was used to investigate whether IRE-1 might be involved in HCPT-induced apoptosis of human fibroblasts. In this study, we found that HCPT-treated fibroblasts failed to recover from ER stress and caused apoptosis by accumulation of GRP78 and activated IRE-1 signals. After the activation of IRE-1, the expression of XBP-1 was also increased. Furthermore, increased expression of the transcription factors CHOP and Bax and decreased expression of Bcl-2 were found in HCPT-treated fibroblasts. To identify whether IRE-1 is involved in HCPT-induced apoptosis of fibroblasts, we successfully knocked down the IRE-1 gene by lentiviral infection. We found that depletion of IRE-1 could lead to decreases in the expression of CHOP and the Bax/Bcl-2 ratio in HCPT-treated fibroblasts. Moreover, the results of Hoechst staining also showed that depletion of IRE-1 could result in prevention of apoptosis after treating fibroblasts with HCPT. Hoechst staining was in conformity with the results of western blot analysis. These results indicated that IRE-1 might play a critical role in HCPT-induced apoptosis of fibroblasts. Likewise, immunohistochemical analysis showed that the expression of IRE-1 in the 1.0 mg/mL and 0.5 mg/mL HPCT-treated groups were higher than those of the control group. Expression of IRE-1 in the 1.0 mg/mL HPCT-treated group was significantly higher than those of the 0.5 mg/mL HPCT-treated group. The results of immunohistochemical coincided with that of western blot analysis. In conclusion, the animal study showed first that HCPT could prevent scar adhesion after knee surgery and that 1 mg/mL HCPT has a better effect than 0.5 mg/mL HCPT. Second, the results of in vitro experiments showed that HCPT could induce apoptosis of human fibroblasts, and the in vivo and in vitro results demonstrated that the IRE-1 signal pathway was involved in HCPT-induced apoptosis of fibroblasts. These results could offer a new strategy to address postoperative intraarticular scar adhesion.
Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant NO. 81271994, 81371971, 81301550 and 81501870).
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway Xiaolei Li a,1, Yu Sun b,1, Hui Chen b, Gengyao Zhu b, Yuan Liang b, Qiang Wang b, Jingcheng Wang b,n, Lianqi Yan b,n a b
Department of Orthopedics, The Second Xiangya Hospital Affiliated with Central South University, Changsha 410011, China Department of Orthopedics, Clinical medical college of Yangzhou University, Subei People’s Hospital of Jiangsu Province, Yangzhou 225001, China
art ic l e i nf o
a b s t r a c t
Article history: Received 10 December 2015 Received in revised form 6 April 2016 Accepted 8 April 2016
Hydroxycamptothecin (HCPT) has been proven to prevent intraarticular scar adhesion, but the mechanism is still unclear. ER stress is known to participate in many diseases, and the IRE-1 signal pathway has been reported in fibrotic diseases. The aim of this study was to illustrate the mechanism of HCPTinduced apoptosis in fibroblasts and the prevention of intraarticular scar adhesion. The effects of HCPT on fibroblasts were determined by CCK-8 assay, Hoechst staining and Western blot. The effect of HCPT on intraarticular scar adhesion was detected by macroscopic evaluation, hydroxyproline content, histological evaluation, fibroblast counting and immunohistochemical analysis. HCPT induced apoptosis of fibroblasts, according to CCK-8 assays, Hoechst staining and Western blot analysis. As the concentration of HCPT increased, the expressions of glucose-regulated protein 78 (GRP78), inositol-requiring kinase1 (IRE-1), C/EBP homologous protein (CHOP) and Bax were all increased, but the expression of Bcl-2 was decreased. Knockdown of IRE-1 alleviated the HCPT-induced apoptosis in our fibroblast model. HCPT could prevent intraarticular scar adhesion, according to the results of macroscopic evaluation, hydroxyproline content, histological evaluation and fibroblast counting in a rabbit model. Immunohistochemical analysis showed that IRE-1 expression increased as the concentration increased. The present study showed that the IRE-1 signal pathway might be involved in HCPT-induced apoptosis of fibroblast and might play a role in preventing intraarticular scar adhesion. & 2016 Elsevier B.V. All rights reserved.
Keywords: Fibroblast apoptosis IRE-1 signal pathway Hydroxycamptothecin Intraarticular scar adhesion
1. Introduction Intraarticular scar adhesion is considered as an important factor after knee surgery in joint department. Stiffness and severe functional impairments, as well as poor clinical outcomes, occur after knee surgery as a result of intraarticular scar adhesion. The etiology of intraarticular scar adhesion is unclear, but fibrous tissue hyperplasia is considered by many authors to have a critical role in the formation of intraarticular scar adhesion. Therefore, various anti-proliferation agents were applied to reduce intraarticular scar adhesion in animal experiments and achieved limited success (Fukui et al., 2001; Hayashi et al., 2004; Sun et al., 2014). n
Corresponding author. E-mail addresses: [email protected] (J. Wang), [email protected] (L. Yan). 1 Xiaolei Li and Yu Sun equally contributed to paper.
Hydroxycamptothecin (HCPT) is a cell cycle-specific DNA topoisomerase I inhibitor agent that shows high activity against the proliferation of many tumor cells (Zhou et al., 2011). HPCT is used as an anti-tumor agent against bladder cancer, gastric cancer, colorectal cancer and has had success in the clinic (Yang et al., 2007; Wang et al., 2007). As an anti-tumor agent, HCPT has been shown to induce the apoptosis of human tendon capsule fibroblasts. Moreover, Yang and Sun have reported that HCPT can minimize epidural scar adhesion by preventing the proliferation of fibroblasts in a rat laminectomy model (Sun et al., 2008; Yang et al., 2011). Both mitochondrial dysfunction and the ER stress-mediated cell death pathway are involved in drug-induced apoptosis in mammalian cells (Yen et al., 2012; Shi et al., 2013). Furthermore, previous studies have shown that caspase-3 and caspase-9 play important roles in HCPT-induced apoptosis in human tendon capsule fibroblasts (Tang et al., 2012). These results showed that endoplasmic reticulum (ER) stress
http://dx.doi.org/10.1016/j.ejphar.2016.04.012 0014-2999/& 2016 Elsevier B.V. All rights reserved.
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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might also be involved in HPCT-induced apoptosis, and further investigation to investigate the possible mechanism of HCPT-induced apoptosis of human fibroblast is warranted. Recently, many studies have shown that ER stress is closely linked to human disease, such as tissue ischemia, Alzheimer's disease, Parkinson's disease and diabetes mellitus (Rozpedek et al., 2015; Yang and Luo, 2015; Harding et al., 2001; Nishitoh et al., 2002). The ER is a multifunctional signaling organelle that plays a critical role in protein folding, handling of misfolded proteins, posttranslational modification and calcium homeostasis (Boyce et al., 2005; Woehlbier et al., 2011). ER stress is triggered by the accumulation of unfolded proteins and altered calcium homeostasis. In response to ER stress, the unfolded protein response (UPR) restores protein homeostasis by suppressing protein translation, inducing ER-related molecular chaperones to promote refolding of unfolded proteins and removing unfolded proteins by activating ER-associated protein degradation (Xu et al., 2005; Merksamer et al., 2010). Inositol-requiring enzyme 1 (IRE-1) was identified as the only sensor of ER stress in yeast and is essential in both animals and plants (Chen and Brandizzi, 2013). Previous results of rat models showed that IRE-1 played a critical role in fibrosis formation in liver cirrhosis (Jiang et al., 2015). The main purpose of this study was to illustrate the effects of IRE-1 on HCPT-induced apoptosis in human fibroblasts and the prevention of intraarticular scar adhesion in rabbit model.
2. Materials and methods The experimental research was approved by the Ethical Committee and Research Committee of Subei Hospital of Jiangsu Province. The primary fibroblast cell line was obtained from GuangZhou Jenino Biotech Co., Ltd. The fibroblasts were cultured in DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin G and 100 U/mL streptomycin in an atmosphere of 5% CO2 at 37 °C. Fibroblast cells of passages 3 and 6 were selected in all of the experiments. 2.1. HCPT treatment HCPT was purchased from Lishizhen pharmaceutical Co., Ltd (Hubei, China). Human fibroblasts were cultured in 24-well plates, 96-well plates or 10-cm dishes overnight. After washing with PBS, human fibroblast cells were treated with HCPT at various concentrations and for various durations. The control group was treated with PBS. After all HCPT and PBS treatments, cells were washed with PBS three times and cultured for subsequent experiments. 2.2. Cell viability test Cell counting Kit-8 (CCK-8) was applied to determine cell viability. The human fibroblast cells were cultured in triplicate in 96well plates. The fibroblast cells were treated with 0.05 μg/mL HCPT, 0.1 μg/mL HCPT, 0.5 μg/mL HCPT, 1 μg/mL HCPT and 4 μg/mL HCPT, upon which treated cells were maintained in DMEM for 12 h, 24 h,36 h, 48 h and 60 h. Cell viability was determined according to the manufacturer's instructions. Briefly, the CCK-8 solution (10 mL) was added to each well for 2 h at 37 °C, and cells that were stained with the CCK-8, were considered viable and were reported as a percentage of the control. 2.3. Hoechst 33342 staining Fibroblasts were plated in 6-well plates (2 mL, 1 106 per well)
and incubated to adhere overnight at 37 °C. Increasing concentrations of HCPT were then added to each well, and the cells were incubated for another 8 h at 37 °C. The fibroblast monolayer was then washed three times with PBS and stained with 5 mg/mL Hoechst for 30 min at 37 °C in the dark. Following staining, the fibroblasts were washed three times with PBS. The morphological features of apoptosis were observed by fluorescence microscopy (Olympus BX 51) with a magnification of 400. 2.4. Western blot analysis HCPT-treated fibroblasts were lysed in RIPA buffer (Beyotime, Hangzhou, China) according to the manufacturer's instructions, and the lysates were centrifuged at 12000g for 15 min. Supernatants were collected for western blot analysis, and the protein concentration was determined using a BCA Protein Assay Kit (Beyotime, Hangzhou, China). Equal amounts (60 μg/lane) of total protein were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and transferred onto PVDF membranes. Membranes were blocked with 5% skim milk in TBST for 2 h at room temperature and subsequently incubated with the primary antibodies overnight. Anti-78-kDa glucose-regulated protein (GRP78), anti-CHOP, anti-phosphoIRE-1a (S724) antibodies, anti-Bax and anti-Bcl-2 antibodies were obtained from Abcam, USA. Anti-inositol-requiring enzyme 1a (IRE-1a) was obtained from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). Anti-poly ADPribose polymerase (PARP), anti-β-actin antibody were purchased from Cell Signaling Technology (Cell Signaling Technology, USA). The membranes were next washed three times in TBST and incubated with secondary antibodies (Santa Cruz Biotechnology, USA) (diluted 1:5 000) for 2 h. The membrane was visualized via an enhanced ECL system (Millipore, USA). 2.5. Gene silencing using lentiviral infection Lentiviral vectors containing the target gene, IRE-1, were purchased from Shanghai Genechem Co. Ltd. (Genechem, China). Lentiviral infection was carried out according to the manufacturer's instructions. Fibroblasts were incubated with the lentiviral vector at an MOI of 20 in the presence of 2 mg/mL polybrene (Gibco, USA) overnight. Subsequently, the medium was removed, and fresh complete medium was added. Following transfection for 48 h, the fibroblast cells were cultured with 2 mg/mL puromycin (Sigma, USA) for 96 h. Stably transfected cells were then used in subsequent experiments, such as HCPT treatment and western blot and so on. 2.6. Animals Thirty-six mature male New Zealand rabbits weighing 3.8 kg were used in the present study. The rabbits were randomly divided into three groups (twelve rabbits in each group): HCPT (1.0 mg/mL) group, HCPT (0.5 mg/mL) group and control group. The rabbits were housed for 1 week to adapt to the condition of the laboratory before the experiment. 2.7. Animal model An animal model of intraarticular adhesion was performed according to our previous studies (Fukui et al., 2000; Yan et al., 2010). All animals were anesthetized by intravenous administration of pentobarbital sodium, and the animals were fixed in the supine position. The knee joint was opened, and the both sides of the femoral condyle were exposed after the hair was shaved and the exposed skin was sterilized with iodophor. Approximately 10 mm 10 mm squares of the cortical bone were removed from
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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Fig. 1. Application of HCPT inhibits cell viability and induces apoptosis in human fibroblasts. (A) Cell viability after various concentrations of HCPT was detected by CCK-8 assay. The results showed that HCPT could inhibit cell viability in a dose-dependent manner. (B) Cell viability after various durations for 1 μg/mL HCPT treatment were also detected by CCK-8 assay. The results showed that 1 μg/mL HCPT could inhibit cell viability in a time-dependent manner. (C) Fibroblast cells with condensed and fragmented nuclei were observed after 1 μg/mL HCPT treatment, according to the result of Hoechst staining. (D) Western blot analysis showed that HPCT could increase the expression of cleaved PARP in a time-dependent manner. (E) The histogram in this panel represents the mean 7 S.E.M of three independent experiments. *P o 0.05 and **Po 0.01 versus control group (0 μg/mL).
both sides of the femoral condyle until the cancerous bone underneath was exposed. The articular cartilage was left intact. 2.8. Application of drugs Cotton pads (15 mm 15 mm) soaked with HCPT 1.0 mg/mL and 0.5 mg/mL were applied on the exposed decorticated area for 10 min in the corresponding group. Then, the soaked cotton pad was removed, and the surgical site was irrigated with saline. In control group, the decorticated area was only irrigated with saline. In all groups, the surgical site was closed in layers with non-absorbable silk sutures, and the surgical knee joint was fixed extraarticular with Kirschner wire (K-wire) for 4 weeks. 2.9. Macroscopic evaluation Six postoperative rabbits were randomly selected from each group for macroscopic evaluation at 4 weeks. They were killed by intravenous administration of a lethal dose of pentobarbital sodium. The joint was reopened by former skin incision. The presence and severity of adhesion was assessed according to the following visual scoring system by a surgeon who was blinded to the treatment: 0: no adhesions; 1: weak, mild, filmy adhesions that can be eliminated by minimal manual traction; 2: moderate adhesions that can be eliminated by manual traction; 3: dense fibrous adhesions that must be surgically removed.
2.10. Hydroxyproline content After macroscopic evaluation, the scar tissue obtained from the surgical sites was used for biochemical HPC analysis. The assay of hydroxyproline in scar tissue (approximately 20 mg) was determined as previously described by Woessner (Yan et al., 2010). The samples were lyophilized, ground and hydrolyzed with 6 mol/ L HCl at 130 °C for 12 h. Then, they were neutralized with 2.5 N NaOH as indicated with methyl red, and 1 mLof chloramine T was added to the hydrolyzed samples and hydroxyproline standards of four known concentrations. After incubation for 20 min at room temperature, 1 mL of hydroxyproline developer (β-dimethylaminobenzaldehyde solution) was added to the samples and the standards. The absorbance of the solution was measured at 558 nm using a spectrophotometer, and the hydroxyproline content of the adhesion tissue was expressed as micrograms per milligram (μg/mg). 2.11. Histological evaluation Histological evaluation was carried out postoperatively in each group four weeks later. Six remaining rabbits in each group were killed by intravenous injection with a lethal dose of pentobarbital sodium solution. The scar was removed from the joint and embedded in paraffin. First, six sections from each group were stained with
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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Fig. 2. HCPT induces ER stress and the IRE-1 pathway. (A) Western blot analysis showed that 1 μg/mL HPCT could increase the expression of CHOP and Bax and decrease the expression of Bcl-2 in a dose-dependent manner. HCPT could induce apoptosis of fibroblasts in a dose-dependent manner. (B–C) HCPT affected ER stress and the IRE-1 pathway in a dose- and time-dependent manner. Fibroblast cells were treated with different concentrations of HCPT for 24 h or treated with 1 μg/mL HCPT for 24 h and cultured for various durations. The expression levels of GRP-78, P-IRE-1, IRE-1, CHOP and β-actin (loading control) were analyzed by western blot. The histograms represent the mean 7 S.E.M of three independent experiments. *Po 0.05 versus control group (0 μg/mL).
hematoxylin-eosin (H&E). The following two parameters were evaluated under the light microscope with the different magnifications: the intraarticular scar adhesion ( 200) and fibroblasts per 100 mm 100 mm counting area ( 400). Second, six sections of each group were stained with Masson Trichrome. The optical density was evaluated under a light microscope with the magnification of 200. 2.12. Immunohistochemical analysis Six sections of scar tissues were prepared for immunohistochemical analysis of IRE-1. The sections were deparaffinized and rehydrated through gradient ethanol solutions according to a routine avidin–biotin–immunoperoxidase technique. After blocking with goat serum solution for 10 min, these sections were incubated with rabbit-IRE-1 antibody (1:200 dilution as supplied by the manufacturer Abcam) overnight at 4 °C. The sections were then washed in PBS solution, and biotinylated anti-goat lgG was used as secondary antibody for 15 min. The sections were visualized under a light microscope with a magnification of 400
after using 3,3′-diaminobenzidine for 5 min and counterstaining with hematoxylin for 2 min at room temperature. 2.13. Statistical analysis The statistical analysis was performed using SPSS software (version15.0). The data were expressed as the mean 7 S. E. M. ANOVA was used to calculate the significance in hydroxyproline content and fibroblast counting. P-values o0.05 were considered statistically significant in all analyses. 3. Results 3.1. HCPT inhibited the cell viability and induced apoptosis of fibroblasts Human fibroblasts were treated with HCPT at increasing concentrations for 24 h. A Cell Counting Kit-8 (CCK-8) assay demonstrated that HCPT could induce a dramatic loss in the viability of fibroblasts, and cell viability reached a relatively minimal level at 4 μg/mL HCPT. As a result, fibroblasts were treated with HCPT (1 μg/mL) for various durations. CCK-8 assays showed that the cell viability reduction was concomitant with prolonged stimulation. According to the results of
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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Fig. 3. IRE-1 knockdown attenuated the HCPT-induced ER stress and apoptosis in human fibroblasts. (A–B) Human fibroblasts were transduced with IRE-1-mediated lentivirus. Silenced IRE-1 expression was verified by western blot analysis. (C–D) Cell viability and apoptosis in normal and IRE-1-knockdown human fibroblasts were measured by CCK-8 assay and Hoechst staining with 1 μg/mL HCPT treatment for 24 h. After the IRE-1-knockdown in human fibroblasts, cell viability was increased, and the number of apoptotic nuclei was decreased. The data are presented as the mean 7 S.E.M (*Po 0.05). (E–F) The expression levels of the ER stress markers GRP78 and CHOP and the downstream Bcl-2 and Bax were determined by western blot in IRE-1-knockdown and control human fibroblasts after treatment with 1 μg/mL HCPT for 24 h. β-actin was used as the loading control. The histograms in each panel represent the mean 7 S.E.M of three independent experiments. *P o 0.05 versus control group (0 μg/mL).
Table1 The results of intraarticular scar adhesions were evaluated according to the visual scoring system.
which displayed a time dependence with stimulation (Fig. 1C D and E). These results suggest that HCPT could induce apoptosis of human fibroblasts. 3.2. HCPT induced ER stress and activated the IRE-1 pathway
Group
HCPT(1.0 mg/mL) HCPT(0.5 mg/mL) Control
Grade 0
1
2
3
3 1 0
3 3 0
0 2 0
0 0 6
CCK-8 described above, HCPT is an effective inhibitor of fibroblast viability in vitro, and this inhibitory effect is both dose and time dependent (Fig. 1 A and B). Hoechst staining and western blot analysis revealed that fibroblast cells treated with 1 μg/mL HCPT contained condensed and fragmented nuclei and cleaved PARP,
Western blot analysis was used to determine whether ER stress was involved in HCPT-induced apoptosis of fibroblasts. The expression of GRP78 and CHOP, which are inducers of ER stress, was detected (Fig. 2). Likewise, HCPT could decrease antiapoptotic Bcl-2 expression, whereas HCPT increased apoptotic Bax and CHOP expression in a dose-dependent manner. Moreover, the expression of GRP78 increased starting at 0.05 μg/mL HCPT and reached a maximum of 4 μg/mL. IRE-1 was activated from 0.05 μg/mL HCPT and reached a maximum at 4 μg/mL in a dose-dependent manner (Fig. 2 A and B). After treatment with 1 μg/mL HCPT, the GRP78 expression was markedly increased at 3 h after treatment and was sustained for 24 h. Importantly, fibroblasts treated with 1 μg/mL HCPT markedly induced the activation of IRE-1 in a time-dependent manner (Fig. 2C). These data indicated that HCPT induced ER stress and activated the IRE-1 pathway, which might be involved
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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Fig. 4. Hydroxyproline contents of intraarticular scar tissues in the 1 mg/mL HCPT, 0.5 mg/mL HCPT and saline groups. The hydroxyproline content was expressed as μg/mg. *Po 0.05, compared with the hydroxyproline content in the control group. #Po 0.05, compared with the hydroxyproline content in the 0.5 mg/mL HCPT group.
Fig. 6. The fibroblast number in intraarticular scar tissues in the 1 mg/mL HCPT, 0.5 mg/mL HCPT and saline groups. The fibroblast number was expressed as the number/counting area (100 mm 100 mm). *P o0.05, compared with the fibroblast number in the control group. #Po 0.05, compared with the fibroblast number in the 0.5 mg/mL HCPT group.
in apoptosis signaling of human fibroblasts.
3.5. Hydroxyproline contents analysis
3.3. Suppression of HCPT-induced apoptosis of human fibroblast via knockdown of IRE-1
The hydroxyproline contents of the 1.0 mg/mL and 0.5 mg/mL HPCT-treated groups were significantly lower than those of the control group (P o 0.05). Furthermore, the hydroxyproline contents of the 1.0 mg/mL HPCT-treated group were significantly lower than those of the 0.5 mg/mL HPCT-treated group (Po 0.05). The hydroxyproline contents of intraarticular scar tissue in each group are shown in Fig. 4.
Previous results showed that IRE-1 is involved in the expression of CHOP, Bcl-2 and Bax during HCPT-induced ER stress in fibroblast cells. Therefore, we knocked down the IRE-1 gene from fibroblast cells by lentiviral-mediated shRNAs. After successfully silencing the expression of IRE-1 (Fig. 3A and B), fibroblast cells were incubated with 1 μg/mL HCPT as described above. Silencing of IRE-1 resulted in a prominent increase in cell viability according to CCK-8 assays (Fig. 3D) and a decrease in apoptosis according to Hoechst staining (Fig. 3C). Meanwhile, the expression of the ER stress markers (GRP78 and CHOP) and the downstream Bax were decreased and that of Bcl-2 was increased significantly in the IRE-1 silenced group (Fig. 3E and F).
3.4. Macroscopic evaluation of intraarticular adhesion All rabbits recovered without mortality or any sign of wound infection or cutaneous necrosis during the four weeks post operation. After four weeks, macroscopic assessment showed that a significant reduction in the adhesion score was observed in the 1.0 mg/mL and 0.5 mg/mL HPCT-treated groups relative to the controls. In the 1.0 mg/mL HPCT-treated group, filmy membrane-like scar tissue covered the surgical site, which could be dissected easily without bleeding. In the 0.5 mg/mL HCPT group, moderate scar tissue covered the surgical area, which could be dissected by manual traction. In contrast, the control group presented severe thick fibrous tissue around the surgical site, which was dissected with difficulty and was accompanied by bleeding. The results of intraarticular adhesion grading scores according to the visual scoring system are shown in Table 1.
3.6. Histological evaluation In the control group, severe fibrosis and dense fibers with a large population of mature fibroblasts were observed around the surgical site. In the 0.5 mg/mL HPCTtreated group, the surgical site was covered with moderate fibrous adhesion with dense fibroblasts. In contrast, in the 1.0 mg/mL HPCT-treated group, loose scar tissues with fewer fibroblasts were observed around the surgical site. Representative histological images of intraarticular adhesion in rabbits of each group are shown in Fig. 5. 3.7. Effect of 1.0 mg/mL and 0.5 mg/mL HPCT on fibroblasts The fibroblast number in scar tissue from the 1.0 mg/mL and 0.5 mg/mL HPCT groups were significantly less than those of the control group (P o 0.05). Moreover, the fibroblast number of the 1.0 mg/mL HCPT group was significantly lower than that of the 0.5 mg/mL HCPT group (P o 0.05). The fibroblast number in the intraarticular scar tissue of each treatment group is shown in Fig. 6.
3.8. Effect of 1.0 mg/mL HPCT and 0.5 mg/mL HPCT on collagen density The results of Masson's Trichrome staining were in agreement with those of hematoxylin-eosin staining. In the control group, there was dense collagen in the
Fig. 5. Representative histological images of intraarticular adhesion in rabbits treated with 1 mg/mL HCPT, 0.5 mg/mL HCPT or saline. These sections were stained with HE, and the magnification is 200 . In the control group, severe fibrosis and dense fibers with a large population of mature fibroblasts were observed around the surgical site. In the 0.5 mg/mL HPCT-treated group, the surgical site was covered with moderate fibrous adhesion with dense fibroblasts. In contrast, in the 1.0 mg/mL HPCT-treated group, loose scar tissues with fewer fibroblasts were observed around the surgical site.
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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Fig. 7. Collagen density in intraarticular scar tissue in rabbits treated with 1 mg/mL HCPT, 0.5 mg/mL HCPT or saline. These sections were stained with Masson, and the magnification is 200 . The results of Masson's Trichrome staining were agreement with those of hematoxylin-eosin staining. In the control group, there was dense collagen in the intraarticular scar tissue. There was low-density collagen in the intraarticular scar tissue in the 1.0 mg/mL HCPT-treated group, which was decreased compare with that of the 0.5 mg/mL HCPT-treated group.
Fig. 8. The results of expression of IRE-1 in the intraarticular scar tissue treated with 1 mg/mL HCPT, 0.5 mg/mL HCPT and saline were similar to the results of the western blot analysis. The results of IRE-1expression were expressed as mean OD and showed in the histogram. *P o 0.05, compared with the fibroblast number in the control group. #Po 0.05, compared with the fibroblast number in the 0.5 mg/mL HCPT group. intraarticular scar tissue. The collagen density was low in the intraarticular scar tissue from the 1.0 mg/mL HCPT-treated group, which was decreased compared with that of the 0.5 mg/mL HCPT-treated group. The results of collagen density in intraarticular scar tissue in rabbits of each group are shown in Fig. 7.
than the 0.5 mg/mL HPCT-treated group. The results of IRE-1 expression were expressed as mean OD and showed in the histogram. *P o 0.05, compared with the fibroblast number in the control group. #Po 0.05, compared with the fibroblast number in the 0.5 mg/mL HCPT group.
3.9. Immunohistochemical analysis of 1.0 mg/mL and 0.5 mg/mL HPCT on IRE-1 expression
4. Discussion
As shown in Fig. 8, the expression of IRE-1 in the intraarticular scar tissue treated with 1 mg/mL HCPT, 0.5 mg/mL HCPT and saline were similar according to western blot analysis. The expression of IRE-1 in the 1.0 mg/mL and 0.5 mg/mL HPCT-treated groups was higher than that of the control group. Furthermore, the expression of IRE-1 in the 1.0 mg/mL HPCT-treated group was significantly higher
Hydroxycamptothecin is natural alkaloid that is isolated form Camptotheca acuminate. It is widely used to treat cancers with less clinical toxicity and was proven to be an anti-fibrosis agent by previous studies. Previous studies have shown that HCPT could
Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i
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induce apoptosis in various cancer cell, such as lung adenocarcinoma, bladder cancer and colon cancer. In addition, HCPT has also been proven to induce apoptosis of both human tendon capsule and hamster lung fibroblasts (Ping et al., 2006; Yokoo et al., 2007; Zhao et al., 2011). Our previous study showed that topical application of 1 mg/mL HCPT might be the optimal concentration for preventing intraarticular scar adhesion in rabbit models, but the mechanism of HCPT reduction in intraarticular scar adhesion by preventing proliferation and inducing apoptosis in fibroblasts is still unclear. Further experiments are needed to elucidate the precise mechanism (Liang et al., 2014; Yan et al., 2015). In the present study, we found that 0.5 mg/mL and 1.0 mg/mL HCPT could induce fibroblast apoptosis and reduce intraarticular scar adhesion in a rabbit model. Multiple parameters, including the visual scoring system, hydroxyproline content analysis, HE staining, the number of fibroblasts and Masson staining, were applied to evaluate the effect of HCPT on preventing intraarticular scar adhesion in a rabbit model. It is known that fibroblasts can produce collagen and extracellular matrix components in bone defect areas. Uncontrolled production of those may lead to the formation of scar tissue and result in intraarticular adhesion. Furthermore, previous research showed that hydroxyproline accounts for 12.5% of the amino acid content in collagen fiber so that the hydroxyproline content can reflect the formation of collagen in scar tissue (Edwards and O’Brien, 1980). Thus, collagen observation, hydroxyproline content and fibroblast counting could reflect the efficacy of HCPT in preventing intraarticular scar adhesion in a rabbit model. The results of macroscopic evaluation and HE staining showed that there were weak and moderate scar adhesions around the bone defect area in HCPT-treated groups. The results of hydroxyproline content and fibroblast counting were also all significantly lower in the HCPT-treated groups than those in the control group. The results of Masson staining were similar to those of HE staining and showed that little or weak collagen appeared in the bone defect area in the HCPT-treated group. Moreover, the results of the 1 mg/mL HCPT-treated group were also less than those of the 0.5 mg/mL HCPT-treated group. These results indicated that HCPT could prevent scar adhesion after knee surgery and that 1 mg/mL HCPT could have a better effect. In vitro, CCK-8 assays and Hoechst staining were used to elucidate the effect of HCPT on fibroblast proliferation and apoptosis in this study. CCK-8 assays demonstrated that HCPT could inhibit the cell viability of human fibroblasts in a dose-dependent manner and reached minimal cell viability at 4 μg/mL. Moreover, we also found that HCPT could inhibit cell viability in a time-dependent manner, according to CCK-8 assays. Hoechst staining showed that human fibroblasts exhibited condensed and fragmented nuclei when stimulated for 24 h by 1 μg/mL HCPT. The ER is an intracellular organelle and is considered an important site of secretor and membrane protein synthesis, modification and folding into their correct conformations. Furthermore, the ER is responsible for intracellular calcium homeostasis, and ER stress is triggered by imbalances in calcium levels. Cells activate an adaptive mechanism known as the UPR to respond to ER stress. If a cell fails to recover from ER stress, the adaptive response is repressed and apoptosis is triggered by the UPR. The UPR is mediated by three signaling pathways: PKR-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE-1) and activating transcription factor 6 (ATF6) (Shore et al., 2011; Hetz et al., 2012). Previous studies showed that IRE-1 was involved in many different fibrosis-related diseases, and it regulates the proliferation, apoptosis and metabolism of fibroblasts (Mo et al., 2015; Zhao et al., 2014). IRE-1 is one of three ER stress signals and is considered to be a regulator of CHOP transcription, which is a central mediator of ER-stress-induced apoptosis (Chan et al., 2015; Choi
et al., 2015). With the activation of ER stress and accumulation of unfolded proteins, IRE-1 dissociates from GRP/BiP and is permitted to dimerize, which induces splicing of XBP1. The generated spliced XBP1 is a potent transcriptional activator that can induce downstream genes, including those encoding ER chaperones and ERassociated proteins (Lenna et al., 2012; Shi et al., 2013; Coelho et al., 2014). Therefore, western blot analysis was used to investigate whether IRE-1 might be involved in HCPT-induced apoptosis of human fibroblasts. In this study, we found that HCPT-treated fibroblasts failed to recover from ER stress and caused apoptosis by accumulation of GRP78 and activated IRE-1 signals. After the activation of IRE-1, the expression of XBP-1 was also increased. Furthermore, increased expression of the transcription factors CHOP and Bax and decreased expression of Bcl-2 were found in HCPT-treated fibroblasts. To identify whether IRE-1 is involved in HCPT-induced apoptosis of fibroblasts, we successfully knocked down the IRE-1 gene by lentiviral infection. We found that depletion of IRE-1 could lead to decreases in the expression of CHOP and the Bax/Bcl-2 ratio in HCPT-treated fibroblasts. Moreover, the results of Hoechst staining also showed that depletion of IRE-1 could result in prevention of apoptosis after treating fibroblasts with HCPT. Hoechst staining was in conformity with the results of western blot analysis. These results indicated that IRE-1 might play a critical role in HCPT-induced apoptosis of fibroblasts. Likewise, immunohistochemical analysis showed that the expression of IRE-1 in the 1.0 mg/mL and 0.5 mg/mL HPCT-treated groups were higher than those of the control group. Expression of IRE-1 in the 1.0 mg/mL HPCT-treated group was significantly higher than those of the 0.5 mg/mL HPCT-treated group. The results of immunohistochemical coincided with that of western blot analysis. In conclusion, the animal study showed first that HCPT could prevent scar adhesion after knee surgery and that 1 mg/mL HCPT has a better effect than 0.5 mg/mL HCPT. Second, the results of in vitro experiments showed that HCPT could induce apoptosis of human fibroblasts, and the in vivo and in vitro results demonstrated that the IRE-1 signal pathway was involved in HCPT-induced apoptosis of fibroblasts. These results could offer a new strategy to address postoperative intraarticular scar adhesion.
Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant NO. 81271994, 81371971, 81301550 and 81501870).
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Please cite this article as: Li, X., et al., Hydroxycamptothecin induces apoptosis of fibroblasts and prevents intraarticular scar adhesion in rabbits by activating the IRE-1 signal pathway. Eur J Pharmacol (2016), http://dx.doi.org/10.1016/j.ejphar.2016.04.012i