Mol. Cells 2018; 41(5): 401-412
Published online May 10, 2018
https://doi.org/10.14348/molcells.2018.2180
© The Korean Society for Molecular and Cellular Biology
Correspondence to : *Correspondence: luhong@zcmu.edu.cn
Oxymatrine (OMT) often used in treatment for chronic hepatitis B virus infection in clinic. However, OMT?induced liver injury has been reported. In this study, we aim to investigate the possible mechanism of OMT-induced hepatotoxicity in human normal liver cells (L02). Exposed cells to OMT, the cell viability was decreased and apoptosis rate increased, the intracellular markers of oxidative stress were changed. Simultaneously, OMT altered apoptotic related proteins levels, including Bcl-2, Bax and pro-caspase-8/-9/-3. In addition, OMT enhanced the protein levels of endoplasmic reticulum (ER) stress makers (GRP78/Bip, CHOP, and cleaved-Caspase-4) and phosphorylation of c-Jun N-terminal kinase (p-JNK), as well as the mRNA levels of GRP78/Bip, CHOP, caspase-4, and ER stress sensors (IREI, ATF6, and PERK). Pre-treatment with Z-VAD-fmk, JNK inhibitor SP600125 and N-acetyl-l-cysteine (NAC), a ROS scavenger, partly improved the survival rates and restored OMT-induced cellular damage, and reduced caspase-3 cleavage. SP600125 or NAC reduced OMT-induced p-JNK and NAC significantly lowered caspase-4. Furthermore, 4-PBA, the ER stress inhibitor, weakened inhibitory effect of OMT on cells, on the contrary, TM worsen. 4-PBA also reduced the levels of p-JNK and cleaved-caspase-3 proteins. Therefore, OMT-induced injury in L02 cells was related to ROS mediated p-JNK and ER stress induction. Antioxidant, by inhibition of p-JNK or ER stress, may be a feasible method to alleviate OMT-induced liver injury.
Keywords apoptosis, ER stress, hepatotoxicity, JNK, L02 cell, oxymatrine
Oxymatrine (OMT, C15H24N2O2) was a quinolizidine alkaloid extracted from traditional Chinese medicinal herbs such as Sophora flavescens and radix sophorae tonkinensis, which has been used in treating conditions like chronic viral hepatitis (Li et al., 1998), leukopenia caused by tumor radiation and chemotherapy (Ho et al., 2009) and plaque psoriasis (Zhou et al., 2017). As a chemically-defined natural product, some studies about OMT pharmacological effects have been developed, such as anti-prostate and colorectal cancer (Wang et al., 2017; Wu et al., 2015), anti-inflammatory (Sun et al., 2008), anti-cardiac, hepatic and renal fibrosis (Fu et al., 2016; Wang et al., 2016; Zhang et al., 2014), anti-virus (Jiang et al., 2017) and anti-arrhythmic effects (Cao et al., 2010). Apart from many findings of its beneficial activities, it was also reported that OMT worsened clinically relevant liver damage (Gao et al., 2002; Li and Huang, 2011; Tian, 2003).
In our previous work, we have found OMT at a dose of 160 and 320 mg/kg leads to obvious liver injury, which was related to the phosphorylation of c-Jun N-terminal kinase (p-JNK) signaling pathway mediated by tumor necrosis factor alpha (TNF-α) in mice liver. As we all known, JNK has a significant role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways (Dhanasekaran and Reddy, 2008). In human cells, multitudinous apoptotic stimuli, such as reactive oxygen species (ROS) and TNF-α, can trigger the activation of apoptosis signal-regulated kinase (ASK) 1, which eventually resulting to the JNK signalling cascade activation and apoptosis (Dai et al., 2017; Fukumoto et al., 2016). In additional, since the endoplasmic reticulum (ER) is an organelle for synthesis, folding and trafficking for protein, ER homeostasis is absolutely critical in hepatocytes, a major cell type rich in ER content (Han et al., 2016). Perturbation of these processes during different pathological states results in a condition known as ER stress, accompanying induced expression of several unfolded protein response (UPR) markers including Inositol-requiring enzyme 1 (IRE1), protein kinase RNA (PKR)-like endoplasmic reticulumkinase (PERK), activating of the transcription factor (ATF)6, glucose-regulated protein 78 (GRP78/Bip) and C/EBP homology protein (CHOP) (Ron and Walter, 2007). When sustained or unresolved ER stress appeared during the course of acute or chronic liver disease, it may account for hepatocyte dysfunction and death (Ozcan and Tabas, 2012).
Thus this study was designed to carry out the OMT-induced hepatotoxicity test in human normal liver cells (L02). And whether ROS-mediated JNK signaling pathway and ER stress induction or not was explored in L02 cells in response to treatment with OMT.
L02 cells were purchased from the Cell Center of the Chinese Academy of Medical Sciences (China), and the cells were previously derived from the American Type Culture Collection (ATCC, USA). L02 cells were cultured and passed in RPMI 1640 medium (GIBCO, USA) supplemented with 10% fetal calf serum (FCS), 200 U/ml of penicillin sodium and 200 U/mL of streptomycin sulfate at 37°C in a humidified atmosphere of 5% CO2 in air.
OMT presents as a white powder, and its purity is 98%. OMT (lot number: ZY151018) was obtained from Shanghai Puzhen Biotechnology Co. Ltd, and diluted in the culture medium. Z-Val-Ala-Asp (OMe)-fluoromethylketone (Z-VAD-fmk, S7023), a pan-caspase inhibitor and SP600125 (S1460), a JNK inhibitor were obtained from Selleck (Shanghai, China), N-acetylcysteine (NAC, S0077, purity 98%), an antioxidant, was purchased from Beyotime Institute of Biotechnology (China), 4-phenylbutyric acid (4-PBA, Cl0041418, purity 98%), a ER Stress inhibiter was purchased from MACKLIN (China). Tunicamycin (TM, T818567, purity > 98%), a chemical inducer of UPR, was purchased from Solarbio (China).
Methyl thiazolyl tetrazolium (MTT) assay was used to detect the cell survival rate. Briefly, cells were seeded into a 96-well plate at a density of 2 × 104cells/ml, and divided into blank control, normal control and OMT group. Medium containing 6, 12, 18, 24 and 30 mmol/L of OMT was added into each well in a volume of 100 μL and at different time points (8, 16, 24 and 48 h), respectively. Four replicated wells were set up for each group. Then 20 μl MTT solutions (Sigma, Shanghai, China) was added into each well and followed by incubation at 37°C for 4 h. The 150 μl of DMSO was further added to all of the wells after discarding culture medium. The optical density (OD) value was detected using a micro-plate reader (Bio-Rad, USA) at a wavelength of 570 nm. The experiments were performed in triplicate.
Hoechst 33342 staining was assayed morphological observation of nuclear change. Cells were seeded in 6-well plates at a density of 5 × 105 cell/ml, incubated with OMT for 24 h. The cell morphology was observed by Invert/phase contrast microscopy (MOTIC AE2000). Then the culture medium were removed and the cells were incubated with 0.5 mL Hoechst 33342 (Beyotime, China) (Yin et al., 2017) for 30 min at 37°C in a humidified atmosphere of 5% CO2 in air. After washed three times with PBS, the apoptotic cells were visualized using inverted fluorescence microscope (Nikon TE2000, Japan).
TUNEL assay (Dong et al., 2017) was performed on cells using the One Step TUNEL Apoptosis Assay Kit (Beyotime, China) according to the manufacturer’s instructions. After OMT treatment, cells were fixed with 4% paraformaldehyde for 0.5 h and then stained with TUNEL reaction mixture for 1 h at 37°C in the dark. The condensed or fragmented nuclei of apoptotic cells were observed using fluorescence microscopy at 200× magnification.
The extent of apoptosis was measured through Annexin V-FITC apoptosis detection kit (Beyotime, China) as described in the manufacturer’s instructions. After exposure to different concentrations of OMT for 24 h, cells were collected, washed twice with PBS, centrifuged for 5min, then gently resuspended in annexin V binding buffer (195 μl) and incubated with Annexin V-FITC (5 μl) and PI (10 μl) in dark for 15 min and analyzed by flow cytometry (BD, FACS Calibur, USA) using the Flo Max software.
Fluorescent dye 2, 7-dichlorofluorescein-diacetate (DCFH-DA, Beyotime, China) was used to detect the intracellular ROS (Li et al., 2016). After exposed cells to OMT, Cells were washed twice and loaded with DCFH-DA (10 μmol/L) and incubated at 37°C for 20 min. The fluorescence of DCFH was measured on inverted fluorescence microscope at an excitation of 485 nm and emission of 520 nm.
The proteins from cells were collected after treated with OMT. The protein concentration was determined using the bicinchoninic acid protein assay kit (Beyotime, China). Cell homogenates were used for the determination of SOD and Lipid Peroxidation MDA levels by using a commercial kit (Beyotime, China). All procedures complied with the manufacturer’s instructions. The results were corrected for their protein content.
Total RNA was extracted from cells using the RNAprep Pure Cell/Bacteria Kit (Tiangen, China). Reverse transcription of total RNA was carried out in a final volume of 20 μl, containing the following reagents (Tiangen, China): 5 × FastKing-RT SuperMix (4 μl) and RNase-free water. The reaction mixtures were then incubated at 42°C (15 min) and 95°C (3 min), and the cDNA samples were obtained. For PCR amplification, primers were designed. GAPDH sequences: 5′-ACAACTTT GGTATCGTGGAAGG-3′ (forward primer) and 5′-GCCATCAC GCCACAGTTTC-3′ (reverse primer); GRP78: 5′-CATCACGCC GTCCTATGTCG-3′ (forward primer) and 5′-CGTCAAAGACC GTGTTCTCG-3′ (reverse primer); CHOP: 5′-GGAAACAGAG TGGTCATTCCC-3′ (forward primer) and 5′-CTGCTTGAGCCG TTCATTCTC-3′ (reverse primer); Caspase-4: 5′-CAAGAGAAG CAACGTATGGCA-3′ (forward primer) and 5′-AGGCAGATG GTCAAACTCTGTA-3′ (reverse primer); PERK: 5′-ACGATGAGA CAGAGTTGCGAC-3′ (forward primer) and 5′-ATCCAAGGC AGCAATTCTCCC-3′ (reverse primer); IRE1:5′-CATCCCCATGC CGAAGTTCA-3′ (forward primer) and 5′-CTGCTTCTCTCCGG TCAGGA-3′ (reverse primer); ATF6:5′-TCCTCGGTCAGTGGA CTCTTA-3′ (forward primer) and 5′-CTTGGGCTGAATTGAAG GTTTTG-3′ (reverse primer). qPCR amplifications were carried out on a ABI 7500 Fast-time PCR system (Applied Bio-systems, USA) using SuperReal PreMix Color (SYBR Green) (Tiangen, China) containing: 2× SuperReal Color PreMix, 50× ROX Reference Dye and RNase-Free ddH2O, and using pre-optimized amplification conditions. Melting curve data were analyzed to determine PCR specificity. Relative fold expressions were analyzed using the 2−ΔΔCt method and GAPDH Ct values as internal reference in each sample.
Cells were lysed using RIPA Lysis Buffer. Total protein samples were denatured and loaded on a SDS-polyacrylamide gel for electrophoresis, and transferred onto PVDF transfer membranes (Millipore, USA). Membranes were blocked and then incubated overnight at 4°C with primary antibodies: Bax Rabbit mAb (#5023,CST), Bcl-2 Rabbit mAb (#4223, CST), pro-Caspase-9 Rabbit mAb (ab32068, abcam), Caspase-8 Rabbit mAb (#AF6442, Affinity), Caspase-3 (8G10) Rabbit mAb (#9665, CST), Phosphor-SAPK/JNK Rabbit mAb (#4668, CST), CHOP (L63F7) Mouse mAb (#2895, CST), Bip (C50B12) Rabbit mAb (#3177, CST), Caspase-4 Antibody (#4405, CST), β–action (C4) (sc-47778, Santa Cruz) at a 1:1,000 dilution in blocking solution. After three washings in TBST for each 10 min, the membranes were incubated with secondary horseradish peroxidase-conjugated goat anti-mouse/rabbit immunoglobulin G. Then the proteins were detected using an enhanced chemiluminescence detection kit. The images were obtained using Mini-PROTEAN gel imaging system (Bio-rad, USA).
All data were presented as means ± SEM. Differences between the control and the treatment groups were determined using a one-way analysis of variance followed by the least significant difference test. The value of
In order to evaluate OMT toxic effect on the L02 cells, the cell vitality was determined by MTT assay. As shown in Fig. 1, cells were treated with various concentrations of OMT at indicated times, the cell viability appeared in an obvious downtrend. Compared with control group, the OMT-treated group had significant statistical difference (
To further illuminate apoptosis, firstly apoptotic cells were detected by TUNEL analysis. Green fluorescence intensity that labeled apoptotic cells were exhibited markedly increased after OMT treatment for 24 h (Fig. 2C). The cell apoptosis rates were also detected by FCM analysis. The apoptosis rates had the tendency of increasing in a dose-dependent manner (
However, whether or not direct inhibition of Caspase activity protects against the injury was not tested. To address this potential issue, Z-VAD-fmk, a pan-caspase inhibitor, was used. After 24 h of incubation with OMT and Z-VAD-fmk, compare with the OMT group, the cell ability was elevated, increased totally approximately by 15% (Fig. 4A), but it remained statistically significant compared with the control group (
In order to determine whether OMT-induced apoptosis was related to oxidative stress in cells, we evaluated the intracellular ROS, MDA contents and SOD activity. Results are shown in Fig. 5, in comparison with the control group, the levels of ROS accumulated in OMT (12 and 18 mmol/L)-treated cells (
To investigate whether oxidative stress was accompanying with the activation of the JNK and ER stress pathways in cells in following OMT treatment, the expression of JNK phosphorylation and several markers of ER stress were detected by Western blotting. Compared with the control group, as shown in Fig. 6, when cells were treated with 0, 16, 12, 18 mmol/L of OMT for 24 h or 10 mmol/L OMT at 12, 24, 48 h, compared with the control group, OMT stimulated the phosphorylation of JNK expression (
To further to the ER stress involved in OMT-induced cell death, we detected the mRNA levels of ER stress makers including GRP78/Bip, CHOP and Caspase 4 as well as three ER stress-signaling molecules PERK, IREI and ATF6.As shown in Fig. 7, With OMT concentration enrichment, OMT dramatically elevated the levels of GRP78/Bip, CHOP and IREI mRNA expression (
To illuminate the relationship between the phosphorylation of JNK and OMT-induced cytotoxicity, we observed the cell viability and cleaved-capase-3 after pre-treatment with SP600125, a JNK inhibitor in OMT-treated cells. Compare with the OMT-treated group, SP600125 increased cell viability (Fig. 8A,
To investigate further, 4-PBA and TM were used to test with OMT exposed to cells respectively, and make sure the role of ER stress involved in OMT-induced cytotoxicity,. Results as shown in Fig. 9A, compare with the OMT group, co-treatment of 4-PBA (2 mmol/L) and OMT elevated the cell viability, increased totally approximately by 20% and alleviated cell morphology damage (Fig. 9B). Whereas, TM (1 μmol/L) had no effect on cell viability induced by OMT. When concentration of TM reached up to 2 μmol/L, compare with the control group, the cell viability was decreased (
OMT, an alkaloid component extracted from the roots of Sophora species, has been shown various pharmacological effects such as anti-inflammatory, anti-fibrosis, and anti-tumor effects and the ability to protect against myocardial damage, etc (Lu et al., 2016b) and exhibits better prospect of clinical development. However the hepatotoxicity induced by OMT is attracting more attention. In order to guarantee its safe application, some in vitro cytotoxicity assays were conducted to explore its safety. L02 cells belong to human normal liver cells, and are anchorage-dependent and epithelioid cells, has became a important cell model to research all kinds of drug hepatotoxicity and damage mechanism. In this study, our findings first show that exposed cells to OMT decreased cell viability, manifested nuclear morphological change and increased apoptosis rate. ROS-caused oxidative stress is a common pathological physiological basis of liver disease with the joint action of cytokines (Ashraf and Sheikh, 2015). Then as we had expected, after treatment with OMT of different concentrations, intracellular ROS levels and MDA contents appeared to go up and SOD activity declined, suggesting the toxic mechanism was related to oxidative stress and apoptosis.
The relative balance between the anti-apoptotic and pro-apoptotic family members influences the susceptibility of cells to a death signal. OMT-induced cytotoxicity accomppyed with the up regulation of apoptotic protein Bax and down regulation of Bcl-2. As such, activation of caspase requires cleavage into smaller subunits under apoptotic conditions. The pro-caspase -3/-8/-9 levels reduced to convert into cleavage in response to OMT treatment, which were similar to that in HL-60 human leukemia cells (Liu et al., 2014). Z-VAD-fmk, a caspase inhibitor with near 100% efficacy in vivo, only partially weaken the cell death and lessen caspase-3 cleavege in OMT-treated group, indicating OMT-Induced injury was a common caspase and non-caspase dependent processing in vitro.
Several prior studies have described that ROS can mediate the sustained activation of JNK pathways (Chye et al., 2014; Schwabe and Brenner, 2006). JNK are important signaling molecules in multiple pathways in liver physiology and diseases (Seki et al., 2012). As we expected, exposed cells to OMT, a marked increase of p-JNK protein levels was observed, which was also consistent with the results
The next step was to clarify whether or not direct inhibition of ROS-dependent JNK signalling pathway protects against the injury during the process. NAC is a source of glutathione and sulfhydryl groups, and a scavenger of free radicals due to its interaction with ROS. SP600125 is a broad spectrum JNK inhibitor for JNK1, JNK2 and JNK3 with IC50 of 40 nmol/L, 40 nmol/L and 90 nmol/L, respectively. In a cell based assay, the recommended concentration of SP600125 is 5–10 μmol/L (Wu et al., 2017). So NAC (1 mmol/L) and SP600125 (10 μmol/L) were applied respectively, the results demonstrated that NAC or SP600125 protect the liver cell from OMT through inhibition of JNK phosphorylation and caspase-3 activation. NAC may be useful in the management of non-paracetamol DILI, however, current available evidence is limited and does not allow for any firm (Chughlay et al., 2016). It is need for further research in this area.
Interestingly, activation of ER stress maker proteins (GRP78/Bip, CHOP) by OMT were not blocked by pre-treatment with NAC or SP600125. Only cleavege of Caspase-4, an ER-resident caspase that has been previously associated with Caspase-3 activation in cases of ER-dependent apoptosis, was reduced by NAC, indicating that scavenging ROS resulted in inhibition of ER stress and partially restored cell viability and that OMT-induced ER stress may be independent of the phosphorylation of JNK. This inductive effect of OMT on liver cells was further proven by using 4-PBA and TM. It has been demonstrated that TM induced pharmacological ER stress and rapidly trans activated pleckstrin homology-like domain, family A, member-3 with a decrease in cell viability, which facilitates liver injury (Han et al., 2016). However, 4-PBA was administered to alleviate drug-induced injury in L02 cells via inhibition of the PERK-ATF4-CHOP pathway, thereby protecting against acute liver failure (Zhang et al., 2016a; 2016b). Our own data indicated that 4-PBA ameliorated OMT-induced cell death due to inhibition of JNK phosphorylation and Capase-3 activation, conversely, TM exacerbated OMT-induced cell growth inhibition.
In conclusion, the present study contributed to the existing knowledge that OMT-induced injury in L02 cells was related to ROS mediated JNK phosphorylation and the contribution of ER Stress. Antioxidant, suppression of JNK phosphorylation or ER stress inhibitor may be a potential detoxicant to liver damage induced by OMT and TCM containing OMT.
Mol. Cells 2018; 41(5): 401-412
Published online May 31, 2018 https://doi.org/10.14348/molcells.2018.2180
Copyright © The Korean Society for Molecular and Cellular Biology.
Li-li Gu1,2, Zhe-lun Shen1,2, Yang-Lei Li1, Yi-Qi Bao1, and Hong Lu1,*
1College of Pharmaceutical science, Zhejiang Chinese Medical University, Hangzhou 310053, Zhejiang Province, China
Correspondence to:*Correspondence: luhong@zcmu.edu.cn
Oxymatrine (OMT) often used in treatment for chronic hepatitis B virus infection in clinic. However, OMT?induced liver injury has been reported. In this study, we aim to investigate the possible mechanism of OMT-induced hepatotoxicity in human normal liver cells (L02). Exposed cells to OMT, the cell viability was decreased and apoptosis rate increased, the intracellular markers of oxidative stress were changed. Simultaneously, OMT altered apoptotic related proteins levels, including Bcl-2, Bax and pro-caspase-8/-9/-3. In addition, OMT enhanced the protein levels of endoplasmic reticulum (ER) stress makers (GRP78/Bip, CHOP, and cleaved-Caspase-4) and phosphorylation of c-Jun N-terminal kinase (p-JNK), as well as the mRNA levels of GRP78/Bip, CHOP, caspase-4, and ER stress sensors (IREI, ATF6, and PERK). Pre-treatment with Z-VAD-fmk, JNK inhibitor SP600125 and N-acetyl-l-cysteine (NAC), a ROS scavenger, partly improved the survival rates and restored OMT-induced cellular damage, and reduced caspase-3 cleavage. SP600125 or NAC reduced OMT-induced p-JNK and NAC significantly lowered caspase-4. Furthermore, 4-PBA, the ER stress inhibitor, weakened inhibitory effect of OMT on cells, on the contrary, TM worsen. 4-PBA also reduced the levels of p-JNK and cleaved-caspase-3 proteins. Therefore, OMT-induced injury in L02 cells was related to ROS mediated p-JNK and ER stress induction. Antioxidant, by inhibition of p-JNK or ER stress, may be a feasible method to alleviate OMT-induced liver injury.
Keywords: apoptosis, ER stress, hepatotoxicity, JNK, L02 cell, oxymatrine
Oxymatrine (OMT, C15H24N2O2) was a quinolizidine alkaloid extracted from traditional Chinese medicinal herbs such as Sophora flavescens and radix sophorae tonkinensis, which has been used in treating conditions like chronic viral hepatitis (Li et al., 1998), leukopenia caused by tumor radiation and chemotherapy (Ho et al., 2009) and plaque psoriasis (Zhou et al., 2017). As a chemically-defined natural product, some studies about OMT pharmacological effects have been developed, such as anti-prostate and colorectal cancer (Wang et al., 2017; Wu et al., 2015), anti-inflammatory (Sun et al., 2008), anti-cardiac, hepatic and renal fibrosis (Fu et al., 2016; Wang et al., 2016; Zhang et al., 2014), anti-virus (Jiang et al., 2017) and anti-arrhythmic effects (Cao et al., 2010). Apart from many findings of its beneficial activities, it was also reported that OMT worsened clinically relevant liver damage (Gao et al., 2002; Li and Huang, 2011; Tian, 2003).
In our previous work, we have found OMT at a dose of 160 and 320 mg/kg leads to obvious liver injury, which was related to the phosphorylation of c-Jun N-terminal kinase (p-JNK) signaling pathway mediated by tumor necrosis factor alpha (TNF-α) in mice liver. As we all known, JNK has a significant role in death receptor-initiated extrinsic as well as mitochondrial intrinsic apoptotic pathways (Dhanasekaran and Reddy, 2008). In human cells, multitudinous apoptotic stimuli, such as reactive oxygen species (ROS) and TNF-α, can trigger the activation of apoptosis signal-regulated kinase (ASK) 1, which eventually resulting to the JNK signalling cascade activation and apoptosis (Dai et al., 2017; Fukumoto et al., 2016). In additional, since the endoplasmic reticulum (ER) is an organelle for synthesis, folding and trafficking for protein, ER homeostasis is absolutely critical in hepatocytes, a major cell type rich in ER content (Han et al., 2016). Perturbation of these processes during different pathological states results in a condition known as ER stress, accompanying induced expression of several unfolded protein response (UPR) markers including Inositol-requiring enzyme 1 (IRE1), protein kinase RNA (PKR)-like endoplasmic reticulumkinase (PERK), activating of the transcription factor (ATF)6, glucose-regulated protein 78 (GRP78/Bip) and C/EBP homology protein (CHOP) (Ron and Walter, 2007). When sustained or unresolved ER stress appeared during the course of acute or chronic liver disease, it may account for hepatocyte dysfunction and death (Ozcan and Tabas, 2012).
Thus this study was designed to carry out the OMT-induced hepatotoxicity test in human normal liver cells (L02). And whether ROS-mediated JNK signaling pathway and ER stress induction or not was explored in L02 cells in response to treatment with OMT.
L02 cells were purchased from the Cell Center of the Chinese Academy of Medical Sciences (China), and the cells were previously derived from the American Type Culture Collection (ATCC, USA). L02 cells were cultured and passed in RPMI 1640 medium (GIBCO, USA) supplemented with 10% fetal calf serum (FCS), 200 U/ml of penicillin sodium and 200 U/mL of streptomycin sulfate at 37°C in a humidified atmosphere of 5% CO2 in air.
OMT presents as a white powder, and its purity is 98%. OMT (lot number: ZY151018) was obtained from Shanghai Puzhen Biotechnology Co. Ltd, and diluted in the culture medium. Z-Val-Ala-Asp (OMe)-fluoromethylketone (Z-VAD-fmk, S7023), a pan-caspase inhibitor and SP600125 (S1460), a JNK inhibitor were obtained from Selleck (Shanghai, China), N-acetylcysteine (NAC, S0077, purity 98%), an antioxidant, was purchased from Beyotime Institute of Biotechnology (China), 4-phenylbutyric acid (4-PBA, Cl0041418, purity 98%), a ER Stress inhibiter was purchased from MACKLIN (China). Tunicamycin (TM, T818567, purity > 98%), a chemical inducer of UPR, was purchased from Solarbio (China).
Methyl thiazolyl tetrazolium (MTT) assay was used to detect the cell survival rate. Briefly, cells were seeded into a 96-well plate at a density of 2 × 104cells/ml, and divided into blank control, normal control and OMT group. Medium containing 6, 12, 18, 24 and 30 mmol/L of OMT was added into each well in a volume of 100 μL and at different time points (8, 16, 24 and 48 h), respectively. Four replicated wells were set up for each group. Then 20 μl MTT solutions (Sigma, Shanghai, China) was added into each well and followed by incubation at 37°C for 4 h. The 150 μl of DMSO was further added to all of the wells after discarding culture medium. The optical density (OD) value was detected using a micro-plate reader (Bio-Rad, USA) at a wavelength of 570 nm. The experiments were performed in triplicate.
Hoechst 33342 staining was assayed morphological observation of nuclear change. Cells were seeded in 6-well plates at a density of 5 × 105 cell/ml, incubated with OMT for 24 h. The cell morphology was observed by Invert/phase contrast microscopy (MOTIC AE2000). Then the culture medium were removed and the cells were incubated with 0.5 mL Hoechst 33342 (Beyotime, China) (Yin et al., 2017) for 30 min at 37°C in a humidified atmosphere of 5% CO2 in air. After washed three times with PBS, the apoptotic cells were visualized using inverted fluorescence microscope (Nikon TE2000, Japan).
TUNEL assay (Dong et al., 2017) was performed on cells using the One Step TUNEL Apoptosis Assay Kit (Beyotime, China) according to the manufacturer’s instructions. After OMT treatment, cells were fixed with 4% paraformaldehyde for 0.5 h and then stained with TUNEL reaction mixture for 1 h at 37°C in the dark. The condensed or fragmented nuclei of apoptotic cells were observed using fluorescence microscopy at 200× magnification.
The extent of apoptosis was measured through Annexin V-FITC apoptosis detection kit (Beyotime, China) as described in the manufacturer’s instructions. After exposure to different concentrations of OMT for 24 h, cells were collected, washed twice with PBS, centrifuged for 5min, then gently resuspended in annexin V binding buffer (195 μl) and incubated with Annexin V-FITC (5 μl) and PI (10 μl) in dark for 15 min and analyzed by flow cytometry (BD, FACS Calibur, USA) using the Flo Max software.
Fluorescent dye 2, 7-dichlorofluorescein-diacetate (DCFH-DA, Beyotime, China) was used to detect the intracellular ROS (Li et al., 2016). After exposed cells to OMT, Cells were washed twice and loaded with DCFH-DA (10 μmol/L) and incubated at 37°C for 20 min. The fluorescence of DCFH was measured on inverted fluorescence microscope at an excitation of 485 nm and emission of 520 nm.
The proteins from cells were collected after treated with OMT. The protein concentration was determined using the bicinchoninic acid protein assay kit (Beyotime, China). Cell homogenates were used for the determination of SOD and Lipid Peroxidation MDA levels by using a commercial kit (Beyotime, China). All procedures complied with the manufacturer’s instructions. The results were corrected for their protein content.
Total RNA was extracted from cells using the RNAprep Pure Cell/Bacteria Kit (Tiangen, China). Reverse transcription of total RNA was carried out in a final volume of 20 μl, containing the following reagents (Tiangen, China): 5 × FastKing-RT SuperMix (4 μl) and RNase-free water. The reaction mixtures were then incubated at 42°C (15 min) and 95°C (3 min), and the cDNA samples were obtained. For PCR amplification, primers were designed. GAPDH sequences: 5′-ACAACTTT GGTATCGTGGAAGG-3′ (forward primer) and 5′-GCCATCAC GCCACAGTTTC-3′ (reverse primer); GRP78: 5′-CATCACGCC GTCCTATGTCG-3′ (forward primer) and 5′-CGTCAAAGACC GTGTTCTCG-3′ (reverse primer); CHOP: 5′-GGAAACAGAG TGGTCATTCCC-3′ (forward primer) and 5′-CTGCTTGAGCCG TTCATTCTC-3′ (reverse primer); Caspase-4: 5′-CAAGAGAAG CAACGTATGGCA-3′ (forward primer) and 5′-AGGCAGATG GTCAAACTCTGTA-3′ (reverse primer); PERK: 5′-ACGATGAGA CAGAGTTGCGAC-3′ (forward primer) and 5′-ATCCAAGGC AGCAATTCTCCC-3′ (reverse primer); IRE1:5′-CATCCCCATGC CGAAGTTCA-3′ (forward primer) and 5′-CTGCTTCTCTCCGG TCAGGA-3′ (reverse primer); ATF6:5′-TCCTCGGTCAGTGGA CTCTTA-3′ (forward primer) and 5′-CTTGGGCTGAATTGAAG GTTTTG-3′ (reverse primer). qPCR amplifications were carried out on a ABI 7500 Fast-time PCR system (Applied Bio-systems, USA) using SuperReal PreMix Color (SYBR Green) (Tiangen, China) containing: 2× SuperReal Color PreMix, 50× ROX Reference Dye and RNase-Free ddH2O, and using pre-optimized amplification conditions. Melting curve data were analyzed to determine PCR specificity. Relative fold expressions were analyzed using the 2−ΔΔCt method and GAPDH Ct values as internal reference in each sample.
Cells were lysed using RIPA Lysis Buffer. Total protein samples were denatured and loaded on a SDS-polyacrylamide gel for electrophoresis, and transferred onto PVDF transfer membranes (Millipore, USA). Membranes were blocked and then incubated overnight at 4°C with primary antibodies: Bax Rabbit mAb (#5023,CST), Bcl-2 Rabbit mAb (#4223, CST), pro-Caspase-9 Rabbit mAb (ab32068, abcam), Caspase-8 Rabbit mAb (#AF6442, Affinity), Caspase-3 (8G10) Rabbit mAb (#9665, CST), Phosphor-SAPK/JNK Rabbit mAb (#4668, CST), CHOP (L63F7) Mouse mAb (#2895, CST), Bip (C50B12) Rabbit mAb (#3177, CST), Caspase-4 Antibody (#4405, CST), β–action (C4) (sc-47778, Santa Cruz) at a 1:1,000 dilution in blocking solution. After three washings in TBST for each 10 min, the membranes were incubated with secondary horseradish peroxidase-conjugated goat anti-mouse/rabbit immunoglobulin G. Then the proteins were detected using an enhanced chemiluminescence detection kit. The images were obtained using Mini-PROTEAN gel imaging system (Bio-rad, USA).
All data were presented as means ± SEM. Differences between the control and the treatment groups were determined using a one-way analysis of variance followed by the least significant difference test. The value of
In order to evaluate OMT toxic effect on the L02 cells, the cell vitality was determined by MTT assay. As shown in Fig. 1, cells were treated with various concentrations of OMT at indicated times, the cell viability appeared in an obvious downtrend. Compared with control group, the OMT-treated group had significant statistical difference (
To further illuminate apoptosis, firstly apoptotic cells were detected by TUNEL analysis. Green fluorescence intensity that labeled apoptotic cells were exhibited markedly increased after OMT treatment for 24 h (Fig. 2C). The cell apoptosis rates were also detected by FCM analysis. The apoptosis rates had the tendency of increasing in a dose-dependent manner (
However, whether or not direct inhibition of Caspase activity protects against the injury was not tested. To address this potential issue, Z-VAD-fmk, a pan-caspase inhibitor, was used. After 24 h of incubation with OMT and Z-VAD-fmk, compare with the OMT group, the cell ability was elevated, increased totally approximately by 15% (Fig. 4A), but it remained statistically significant compared with the control group (
In order to determine whether OMT-induced apoptosis was related to oxidative stress in cells, we evaluated the intracellular ROS, MDA contents and SOD activity. Results are shown in Fig. 5, in comparison with the control group, the levels of ROS accumulated in OMT (12 and 18 mmol/L)-treated cells (
To investigate whether oxidative stress was accompanying with the activation of the JNK and ER stress pathways in cells in following OMT treatment, the expression of JNK phosphorylation and several markers of ER stress were detected by Western blotting. Compared with the control group, as shown in Fig. 6, when cells were treated with 0, 16, 12, 18 mmol/L of OMT for 24 h or 10 mmol/L OMT at 12, 24, 48 h, compared with the control group, OMT stimulated the phosphorylation of JNK expression (
To further to the ER stress involved in OMT-induced cell death, we detected the mRNA levels of ER stress makers including GRP78/Bip, CHOP and Caspase 4 as well as three ER stress-signaling molecules PERK, IREI and ATF6.As shown in Fig. 7, With OMT concentration enrichment, OMT dramatically elevated the levels of GRP78/Bip, CHOP and IREI mRNA expression (
To illuminate the relationship between the phosphorylation of JNK and OMT-induced cytotoxicity, we observed the cell viability and cleaved-capase-3 after pre-treatment with SP600125, a JNK inhibitor in OMT-treated cells. Compare with the OMT-treated group, SP600125 increased cell viability (Fig. 8A,
To investigate further, 4-PBA and TM were used to test with OMT exposed to cells respectively, and make sure the role of ER stress involved in OMT-induced cytotoxicity,. Results as shown in Fig. 9A, compare with the OMT group, co-treatment of 4-PBA (2 mmol/L) and OMT elevated the cell viability, increased totally approximately by 20% and alleviated cell morphology damage (Fig. 9B). Whereas, TM (1 μmol/L) had no effect on cell viability induced by OMT. When concentration of TM reached up to 2 μmol/L, compare with the control group, the cell viability was decreased (
OMT, an alkaloid component extracted from the roots of Sophora species, has been shown various pharmacological effects such as anti-inflammatory, anti-fibrosis, and anti-tumor effects and the ability to protect against myocardial damage, etc (Lu et al., 2016b) and exhibits better prospect of clinical development. However the hepatotoxicity induced by OMT is attracting more attention. In order to guarantee its safe application, some in vitro cytotoxicity assays were conducted to explore its safety. L02 cells belong to human normal liver cells, and are anchorage-dependent and epithelioid cells, has became a important cell model to research all kinds of drug hepatotoxicity and damage mechanism. In this study, our findings first show that exposed cells to OMT decreased cell viability, manifested nuclear morphological change and increased apoptosis rate. ROS-caused oxidative stress is a common pathological physiological basis of liver disease with the joint action of cytokines (Ashraf and Sheikh, 2015). Then as we had expected, after treatment with OMT of different concentrations, intracellular ROS levels and MDA contents appeared to go up and SOD activity declined, suggesting the toxic mechanism was related to oxidative stress and apoptosis.
The relative balance between the anti-apoptotic and pro-apoptotic family members influences the susceptibility of cells to a death signal. OMT-induced cytotoxicity accomppyed with the up regulation of apoptotic protein Bax and down regulation of Bcl-2. As such, activation of caspase requires cleavage into smaller subunits under apoptotic conditions. The pro-caspase -3/-8/-9 levels reduced to convert into cleavage in response to OMT treatment, which were similar to that in HL-60 human leukemia cells (Liu et al., 2014). Z-VAD-fmk, a caspase inhibitor with near 100% efficacy in vivo, only partially weaken the cell death and lessen caspase-3 cleavege in OMT-treated group, indicating OMT-Induced injury was a common caspase and non-caspase dependent processing in vitro.
Several prior studies have described that ROS can mediate the sustained activation of JNK pathways (Chye et al., 2014; Schwabe and Brenner, 2006). JNK are important signaling molecules in multiple pathways in liver physiology and diseases (Seki et al., 2012). As we expected, exposed cells to OMT, a marked increase of p-JNK protein levels was observed, which was also consistent with the results
The next step was to clarify whether or not direct inhibition of ROS-dependent JNK signalling pathway protects against the injury during the process. NAC is a source of glutathione and sulfhydryl groups, and a scavenger of free radicals due to its interaction with ROS. SP600125 is a broad spectrum JNK inhibitor for JNK1, JNK2 and JNK3 with IC50 of 40 nmol/L, 40 nmol/L and 90 nmol/L, respectively. In a cell based assay, the recommended concentration of SP600125 is 5–10 μmol/L (Wu et al., 2017). So NAC (1 mmol/L) and SP600125 (10 μmol/L) were applied respectively, the results demonstrated that NAC or SP600125 protect the liver cell from OMT through inhibition of JNK phosphorylation and caspase-3 activation. NAC may be useful in the management of non-paracetamol DILI, however, current available evidence is limited and does not allow for any firm (Chughlay et al., 2016). It is need for further research in this area.
Interestingly, activation of ER stress maker proteins (GRP78/Bip, CHOP) by OMT were not blocked by pre-treatment with NAC or SP600125. Only cleavege of Caspase-4, an ER-resident caspase that has been previously associated with Caspase-3 activation in cases of ER-dependent apoptosis, was reduced by NAC, indicating that scavenging ROS resulted in inhibition of ER stress and partially restored cell viability and that OMT-induced ER stress may be independent of the phosphorylation of JNK. This inductive effect of OMT on liver cells was further proven by using 4-PBA and TM. It has been demonstrated that TM induced pharmacological ER stress and rapidly trans activated pleckstrin homology-like domain, family A, member-3 with a decrease in cell viability, which facilitates liver injury (Han et al., 2016). However, 4-PBA was administered to alleviate drug-induced injury in L02 cells via inhibition of the PERK-ATF4-CHOP pathway, thereby protecting against acute liver failure (Zhang et al., 2016a; 2016b). Our own data indicated that 4-PBA ameliorated OMT-induced cell death due to inhibition of JNK phosphorylation and Capase-3 activation, conversely, TM exacerbated OMT-induced cell growth inhibition.
In conclusion, the present study contributed to the existing knowledge that OMT-induced injury in L02 cells was related to ROS mediated JNK phosphorylation and the contribution of ER Stress. Antioxidant, suppression of JNK phosphorylation or ER stress inhibitor may be a potential detoxicant to liver damage induced by OMT and TCM containing OMT.
Jong-Il Shin, Yong-Joon Jeon, Sol Lee, Yoon Gyeong Lee, Ji Beom Kim, and Kyungho Lee
Mol. Cells 2019; 42(3): 252-261 https://doi.org/10.14348/molcells.2019.2440Dae-Hwan Nam, Jung-Hwa Han, Jae Hyang Lim, Kwon Moo Park, and Chang-Hoon Woo
Mol. Cells 2017; 40(7): 457-465 https://doi.org/10.14348/molcells.2017.2296Soojin Hwang, Darae Kim, Gahee Choi, Seon Woo An, Yoon Ki Hong, Yoon Seak Suh, Min Jung Lee,
and Kyoung Sang Cho*