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The effect and underlying mechanism of Timosaponin B-II on RGC-5 necroptosis induced by hydrogen peroxide
- San-Hong Jiang†1,
- Lei Shang†1,
- Li-Xiang Xue2,
- Wei Ding1,
- Shuang Chen1,
- Ruo-Fei Ma3,
- Ju-Fang Huang1Email author and
- Kun Xiong1Email author
© Jiang et al.; licensee BioMed Central Ltd. 2014
Received: 22 August 2014
Accepted: 26 November 2014
Published: 2 December 2014
Necroptosis is an important mode of cell death, which is due to oxidant stress accumulation. Our previous study indicated that oxidant stresses could be reduced by Timosaponin B-II (TBII), a kind of Chinese herb RhizomaAnemarrhenae monomer extraction. We wonder the possible effect of Timosaponin B-II, whether it can protect cells from necroptosis via reducing the oxidant stress, in RGC-5 following hydrogen peroxide (H2O2) insult.
RGC-5 cells were grown in DMEM, the model group was exposed in H2O2 with the concentration of 300 μM, and the experimental group was pre-treated with Timosaponin B-II at different concentrations (1 μM, 10 μM, 100 μM and 1000 μM) for 24 hrs. MTT assay was carried out to measure the cytotoxicity of H2O2, MDA concentration assay was executed to evaluate the degree of oxidative stress, TNF-α ELISA Assay was used to measure the concentration of TNF-α, finally, the degree of necrosis were analyzed using flow cytometry.
We first constructed the cell injury model of necroptosis in RGC-5 upon H2O2 exposure. Morphological observation and MTT assay were used to evaluate the degree of RGC-5 death. MDA assay were carried out to describe the degree of oxidant stress. Annexin V/PI staining was used to detect necroptotic cells pre-treated with or without Timosaponin B-II following H2O2 injury. TNF-α ELISA was carried out to detect the TNF-α accumulation in RGC-5. Upon using Timosaponin B-II with concentration of 100 μM, the percentage of cell viability was increased from 50% to 75%, and the necrosis of cells was reduced from 35% to 20% comparing with H2O2 injury group. Oxidant stress and TNF-α was reduced upon injury which decreased the ratio of RGC-5 necroptosis.
Our study found out that Timosaponin B-II might reduce necroptosis via inhibition of ROS and TNF-α accumulation in RGC-5 following H2O2 injury.
Necroptosis is a novel kind of cell death which has similar morphological features of necrosis [1, 2]. The previous studies pointed out that necroptosis was usually initiated by TNF-α or FasL, etc. and transmitted by certain specific cytokines, which results in the accumulation of oxidative products and eventually leading to necrosis. However, it can be blocked by necrostatin-1 (Nec-1, a specific inhibitor of necroptosis) . Necroptosis existed in different cell types, such as epidermal keratinocytes , Jurkat T cells , L929 cells [5, 6], HepG2 cells , hippocampal neurons , cortical neurons  and photoreceptor cells , etc. upon injury. Furthermore, Rosenbaum, et al. have found a large number of necrotic cells exist in rat retinal ganglion cell layer by PI staining at the early stage of acute high intra-ocular pressure (aHIOP) . The numbers of PI-positive neuron decreased, and the "b" wave was partially restored in flash ERG with Nec-1 pretreatment. Dvoriantchikova’s recent works showed that retinal ganglion cells (RGCs) necroptosis promoted in aHIOP-induced retinal damage , which has the similar finding with our previous work . Additionally, our recent studies have also indicated that necroptosis occurs in retinal ganglion cells-5 (RGC-5) at an early stage following elevated hydrostatic pressure (EHP) in vitro or hydrogen peroxide (H2O2) treatment (our unpublished data) detected by flow cytometry (a typical way to monitor necroptosis) .
The mouse retinal ganglion cell line (RGC-5) was contributed by Department of Ophthalmology, Second Hospital of Ji Lin University, China . RGC-5 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM, HyClone Laboratories, Inc. UT, USA) and supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, Inc. UT, USA), 100 U/ml of penicillin and 100 μg/ml of streptomycin (HyClone Laboratories, Inc. UT, USA). The RGC-5 cells used in the experiment were with 2-3 passages post-thawed to minimize the variability in the assays based on our observations. The density of RGC-5 cells was around 80% in 6 ml culture media in 50 ml flask before insults (H2O2 treatment).
The powder of Timosaponin B-II was provided by Professor Wan-Sheng Chen from Department of Pharmacology, School of Pharmacology, Second Military Medical University, Shanghai, China [29, 30] and its purity is above 98%. Timosaponin B-II was dissolved in sterile normal saline (NS) at 2 mM in room temperature (24°C), H2O2 (Sigma-Aldrich, MO, USA) with 0.01 M PBS at 3 mM in store to yield a low concentration working solutions.
Cell model construction and drug treatment
Cells were equally divided into three groups randomly. The normal control group (CTL), the model group (H2O2, 300 μM), and the experimental group (H2O2 + Timosaponin B-II). The experimental group was pre-treated with Timosaponin B-II at different concentrations (1 μM, 10 μM, 100 μM and 1000 μM) for 24 hrs. No drugs were exposed to the normal control or the model group. After that, we used H2O2 of 300 μM for 12 hrs to get cell insult and randomly selected three in each group for morphological studies while the remaining cells were used for biochemistry studies. For morphological study, the pictures of the attached cells were captured using inverted microscope in 10 × objective (Olympus, CKX41, Tokyo, Japan). We have captured ten pictures at least in each group, and selected the typical graphics to illustrate.
Cytotoxicity of H2O2 model group and Timosaponin B-II pretreated group (1 μM, 10 μM, 100 μM and 1000 μM) were assessed in RGC-5 cells by measuring the amount of insoluble formazan formed in live cells based on the reduction of 3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) salt (Nanjing Jian-Cheng Bio-engineering Institute, Jiangsu, China) according to the manufacturer's protocol. The cells were seeded in 96-well plates with a density of 104 cells/well incubated for 24 hrs at 37°C and 5% CO2. The cells were pretreated with different concentrations of Timosaponin B-II before H2O2 insult or H2O2 used alone and PBS as a negative control. Within 24 hrs pre-treatment of Timosaponin B-II, 50 μl of MTT labeling reagent (2 μg/ml) was added to each well. The plates were incubated at 37°C in a humidified atmosphere with 5% CO2 for 4 hrs. Thereafter, 100 μl of the solubilization solution was added to each well and followed by incubation overnight at 37°C to dissolve formazan crystals. Absorbance was ultimately read using an ELISA plate reader (Bio-tek, ELx800, IL, USA) at a wavelength of 570 nm. Where, H2O2 model group and Timosaponin B-II pretreated group are mean absorbance of treated cells and negative control, respectively.
MDA concentration assay
MDA levels in RGC-5 extractions were assayed using a commercial kit according to manufacturer’s instructions (Nanjing Jian-Cheng Biotechnical Co., Jiangsu, China) as in our previous study , the standard reference substance named tetraethoxypropane were used in 10 nmol/ml. Equal quantities (100 μg) of protein were loaded in each well and each analysis performed in duplicate.
TNF-α ELISA assay
A RGC-5 TNF-α concentration assay was performed using a commercial TNF-α ELISA kit (Invitrogen, CA, USA). The detailed processes were conducted according to the manual included in the kit. Positive control: the antibody tested in the kit was replaced by mouse TNF-α (provided in assay, concentration: 720 ng/L). Equal quantities of protein (80 μg) were analyzed in every tested well and the measurements were carried out by Bio-tek microplate-reader (ELx800, IL, USA). The percentage of TNF-α concentration in normal control group was set as 100%. All experiments were repeated at least twice.
The cells attached to the flasks were trypsinized followed by a gentle wash. The experimental group was pre-treated with Timosaponin B-II at 100 μM. The model group was treated with H2O2 at 300 μM. Resuspended the cells in 200 μl of 1× binding buffer, and then added 5 μl of 20 μg/ml Annexin V and 10 μl of 50 mg/ml PI, incubated at RT for 15 mins in the dark. After the cells were washed and analyzed by FACS Calibur (Becton, Dickinson Company, NJ, USA). The percentages of cells in each quadrant were analyzed using ModFit software (Verity Software House Topsham, NJ, USA). Statistical results of flow cytometry were conducted by calculating the PI+ cells numbers. All the results were repeated three times.
Figure panels were assembled by using Photoshop CC (Adobe, CA, USA). The data were analyzed by using SPSS 19.0 (SPSS, IL, USA). One-way analysis of variance (one-way ANOVA) was performed to test differences in average value between groups. All results were presented as mean ± SD. A value of p < 0.05 was considered statistically significant.
Timosaponin B-II could protect RGC-5 from H2O2 injury
Timosaponin B-II could increase RGC-5 viability upon H2O2 injury
Timosaponin B-II reduced MDA production in RGC-5
Timosaponin B-II inhibited TNF-α production in RGC-5
Timosaponin B-II may decrease the rate of RGC-5 necrosis
At present, mechanism of cell death is one of the hotspot in the field of life science research. As far as we are concerned, compared to apoptosis and autophagy, the researchers did not pay enough attention to necrosis due to the traditional viewpoint that it cannot be modulated and intervened. Necroptosis is the latest cell death mode which was conceptualized in last century in mid-1980s . Since then, it started to catch more and more attention [32–34]. We speculate that damaged cells could be rescued if necroptosis can be intervened at an early stage, and it will also help us find a better strategy for rational interventional therapy in the future.
Chinese herb has a wealth of resources from natural materials with lower side effects, more economical and other advantages, etc. However, it also has the disadvantages such as its complicated composition which cannot be identified easily. Timosaponin B-II is a monomer which is extracted from Chinese herb rhizomaanemarrhenae, it has multiple pharmacological effects, including anti-inflammatory [21, 35], anti-diabetic , anti-oxidative stress [23, 26, 27], anti-senile dementia , learning ability or memory improvement  and neuro-protection  or anti-apoptosis effect in human umbilical vein endothelial cells , etc. Our MTT assay results showed that viability of RGC-5 in model group (H2O2 treatment) was significantly decreased, while RGC-5 was pre-incubated by Timosaponin B-II (1 μM, 10 μM and 100 μM groups) for 24 hrs, RGC-5 proliferation increased gradually in a dose-dependent manner. The RGC-5 proliferation of 100 μM treatment group reached a peak, but it significantly decreased in 1000 μM treatment group. The morphological results suggested that the number of RGC-5 decreased remarkably in model group, the survival of cells tends to be similar as MTT when pretreated with Timosaponin B-II. Taken all together, it indicated that Timosaponin B-II played a protective role on RGC-5 in H2O2-induced damage under a certain concentration. In addition, our results in flow cytometry showed that the number of RGC-5 cell necrosis significantly reduced in 100 μM Timosaponin B-II pretreated group, which suggested that Timosaponin B-II may be partly involved in inhibiting RGC-5 necroptosis under H2O2 exposed conditions. Moreover, our results on oxidation products measurement also showed that Timosaponin B-II could significantly decrease the accumulation of MDA in RGC-5 in certain range of concentrations. Furthermore, we had observed that TNF-α was also inhibited in Timosaponin B-II pretreatment group. Considering one of the most important pathways in necroptosis (TNF-α induced necroptosis pathway initiate-----necroptosis related molecular modulation-----ROS accumulation------cell necrosis [39, 40]), we speculated that Timosaponin B-II might inhibit the RGC-5 necroptosis by reducing oxidation products and TNF-α level.
Finally, it is worthy of noting that our experiments revealed the following two questions. Firstly, although Timosaponin B-II can reduce necroptosis by resisting oxidative stress and reduce pro-inflammatory molecule, but it is impossible to recover to normal level, which indicates its limited function as monomer herbal extraction, it also indirectly supports the compatibility usage of Chinese herbal drugs. Secondly, our results showed that higher concentrations of Timosaponin B-II (e.g. 1000 μM) could be toxic. Frank’s research showed Timosaponin B-II and Timosaponin A-III have the similar structure (such as steroid), but Timosaponin B-II presents one more glycosyl . Timosaponin B-II has the trend to convert into Timosaponin A-III. However higher concentrations of Timosaponin A-III has been regarded as highly cytotoxic [41–43] and could cause cell death instead of protective effect. Therefore, we would consider discarding this concentration (1000 μM) in future studies. Until now, the molecule of regulation mechanism for cellular necroptosis upon injury was concerned, including receptor interacting proteins (RIPs) [8, 10, 12, 13], calpains [14, 44], CDGSH iron-sulfur domain-containing protein 1 (CISD1)  or ubiquitin C-terminal hydrolase (UCH-L1) , etc. Therefore, whether the possible pathway of Timosaponin B-II neuro-protection may mediate by these molecules mentioned above needs further investigation.
Timosaponin B-II with limited concentrations can partially protect RGC-5 from H2O2 induced-necroptosis, which may related to TNF-α and ROS accumulation inhibition.
This work is supported by National Natural Science Foundation of China (#81371011), Wu Jie-Ping Medical Foundation of the Minister of Health of China (320.6750.14118), Natural Science Foundation of Hunan Province (#2015JJ2187) and Young Teachers Training Program of Normal University of Hunan Province to KX. We are grateful for the provision of Timosaponin B-II from Professor Wan-Sheng Chen in Department of Pharmacology, School of Pharmacology, Second Military Medical University, Shanghai, China.
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