Int J Clin Exp Med 2019;12(7):8342-8349www.ijcem.com /ISSN:1940-5901/IJCEM0090332

Original ArticleProtective effect of Xiaoyao-jieyu-san on oxidative damage induced by hydrogen peroxide in PC12 cells

Changde Wang1*, Chunlan Wu1*, Xiao Cheng1, Ziyuan Dong1, Bangjiang Fang2

1Department of Encephalopathy, Shanghai TCM-Integrated Hospital Affiliated to Shanghai University of TCM, Shanghai 200082, China; 2Department of Emergency, Longhua Hospital Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China. *Equal contributors.

Received December 23, 2018; Accepted April 8, 2019; Epub July 15, 2019; Published July 30, 2019

Abstract: This study aimed to evaluate the protective effect of Xiaoyao-jieyu-san (XYJY) on oxidative damage in-duced by H2O2 in PC12 cells. Oxidatively damaged PC12 cells were induced by H2O2 (100 μmol/l). CCK-8 assays were carried out to determine the effects of XYJY on cell viability of H2O2 induced PC12 cells. Subsequently, flow cytometry analysis was used to evaluate apoptosis and ROS levels of H2O2 induced PC12 cells after treatment with XYJY. Furthermore, malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) were de-termined by using commercial kits, and expression of caspase-3, AKT, p-AKT, eNOS, and p-eNOS were evaluated by using Western blotting. The results reveal that XYJY increased cell viability of H2O2 induced PC12 cells during 50 to 800 μg/ml, and XYJY dose-dependently reduced apoptotic cells and ROS levels in H2O2 induced PC12 cells. Furthermore, XYJY treatments (100, 200, and 400 μg/ml) significantly reduced the levels of MDA whereas they increased the levels of SOD and GSH-PX in H2O2 induced PC12 cells. In addition, XYJY treatments (200 and 400 μg/ml) significantly up-regulated the expression of p-AKT and p-eNOS in H2O2 induced PC12 cells, whereas they down-regulated caspase-3. In conclusion, the results presented here suggest that XYJY has a protective effect against oxidative damage in PC12 cells mediated by regulation of AKT/eNOS signaling pathway. The present study could be beneficial for interpretation of the possible molecular mechanisms corresponding to the curative effects of XYJY against stroke and its related diseases.

Keywords: Xiaoyao-jieyu-san, oxidative damage, PC12 cells, stroke, AKT/eNOS

Introduction

Stroke is a worldwide epidemic severe life-threatening disease with high fatality and dis-ability rate [1], among which ischemic stroke patients constitute most of the cases of stroke patients [2]. Furthermore, due to lack of blood flow in the brain after ischemic stroke and the following ischemia-reperfusion, it commonly leads to damage of neurons and the related connected neural circuits and even unexpected behavioral impairments, resulting in heavy bur-den to stroke patients and their families [3, 4]. Consequently, it is important and necessary to protect the neurons in ischemic stroke.

Increasing evidence has suggested that herbal medicines are natural precious resource for finding potential drugs for human being and tra-ditional Chinese medicines (TCMs) have been

used for curing various diseases for thousands years [5, 6]. Xiaoyao-jieyu-san (XYJY) is an em- pirical TCM prescription for treating stoke and its related diseases for decades [7, 8]. The XYJY is derived from the famous Chinese medicinal formulae, and consists of 11 herbal medicines, including whole plant of Bupleurum chinense (15 g), rhizome of Cyperus rotundus (12 g), radix of Curcuma rcenyujin (12 g), radix of Angelica sinensis (15 g), radix of Cynanchum otophyllum (30 g), fructus of Citrus aurantium (15 g), cortex of Albizia julibrissin (15 g), rhi-zome of Acorus tatarinowii (12 g), caulis of Fallopia multiflora (30 g), semen of Ziziphus jujuba Mill. var. spinosa (15 g), and rhizome of Atractylodis macrocephalae (15 g). In prior work, XYJY was found to possess significant curative effects on post-stroke depression via regulation of brain derived neurotrophic factor (BDNF), and the constituents of XYJY mainly

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includes paeoniflorin, imperatorin, naringin, ar- nesene, 2,3,5,4’-tetrahydroxyl-diphenylethyle- ne-2-O-glucoside, kaempferol-3-O-rutinoside, quercetin, hesperidin, cycloastragenol, and at- ractylenolide III [7-9]. In the present study, the protective effect of Xiaoyao-jieyu-san (XYJY) on the oxidative damage induced by hydrogen per-oxide (H2O2) was evaluated in rat pheochromo-cytoma derived cell line (PC12) cells. This work could be beneficial for interpretation of the molecular mechanisms corresponding to the curative effects of XYJY against stroke.

Materials and methods

Chemicals and reagents

Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco. Ltd. Co. (USA); Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Biochem (Shanghai, China). Trypsinase, acrylamide, so- dium lauryl sulfate (SDS), penicillin, streptomy-cin and phosphate buffered saline (PBS) were purchased from JRDun Biotech. (Shanghai, China). Malondialdehyde (MDA), reduced gluta-thione (GSH) and superoxide dismutase (SOD) assay kits were supplied by Nanjing Jiangcheng Bioengineering Institute (Nanjing, China). An- nexin V/FITC kit and reactive oxygen species (ROS) testing kit were purchased from the BD Biosciences (Shanghai, China). Caspase-3, AKT, p-AKT, eNOS, p-eNOS, and GAPDH anti-bodies were purchased from Abcam Biotech. (Shanghai, China). BCA protein assay kit, goat-anti-rabbit/rat horseradish-peroxidase-conju-gated secondary antibodies were obtained from Beyotime Biotechnology (Shanghai, Chi- na).

Cell culture

Rat pheochromocytoma derived cell line (PC12) was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). PC12 Cells were cultured in DMEM medium contain-ing 10% heat-inactivated FBS, 100 U/mL peni-cillin and 100 mg/mL streptomycin at 37°C in 5% CO2 atmosphere.

Preparation of XYJY extracts

The XYJY is supplied by the pharmacy depart-ment of our hospital. In brief, all the 11 herbal medicines were decocted with 8 times water

(v/w) for 1.5 hours. The filtrates concentrated by using rotary evaporators under 60°C, and the fluid extracts of XYJY were afforded (yields of XYJY extracts were calculated as approxi-mately 7.81%) [9].

CCK-8 assay

Cells (5 × 104/100 μl) were plated in the 96-well plates for growth of 24 hours. Then, XYJY at the finally concentrations of 50, 100, 200, 400 and 800 μg/ml was added and cultured for 0, 24, 48, and 72 hours at 37°C with the presence of H2O2 (100 μmol/l). Consequently, 100 μl serum free DMEM containing 10% CCK-8 reagents (v/v) were consequently added in each well, then cells were cultured for 1 hour at 37°C. Finally, optical density values (OD) were read under 450 nm by micro-plate reader. The exper-iment was repeated three times.

Apoptosis and ROS assays by flow cytometry analysis

Cells were plated in the 24-well plates for gr- owth of 24 hours. Then, XYJY at the finally con-centrations of 100, 200, and 400 μg/ml was added and cultured for 24 hours at 37°C with the presence of H2O2 (100 μmol/l). Then, cells were harvested, and then cells (5 × 104) were washed using PBS and stained by the Annexin V/FITC kit, and cell apoptosis was detected by the flow cytometry (FCM) assay on a FACS Calibur flow cytometer (BD Bioscience, USA). In addition, DCFH-DA ROS kit was used to deter-mine the intracellular ROS level by the flow cytometry (FCM) assay. The experiment was repeated three times.

Antioxidant indexes analysis

Cells were plated in the 24-well plates for gr- owth of 24 hours. Then, XYJY at the finally con-centrations of 100, 200, and 400 μg/ml was added and cultured for 24 hours at 37°C with the presence of H2O2 (100 μmol/l). Then, cells were harvested and MDA, GSH, and SOD were determined by commercial kits following the manufacture’s instruction.

Western blot assay

Total proteins of the PC12 cells were extracted. Subsequently, 35 μg total proteins were sepa-rated by sodium dodecylsulfate-polyacrylamide

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ring the concentration of 200 μg/ml to 400 μg/ml at the time points designed, including 24, 48, and 72 hours (p<0.01). Consequ- ently, the results indicate that XYJY has a potential protective effect against the H2O2 induced injury in PC12 cells.

Effects of XYJY on H2O2 induced apoptosis of PC12 cells

In the present study, the flow cytometry assay with Annexin V-FITC/PI staining was carried out to evaluate the effects of XYJY on H2O2 induced apoptosis of PC12

gel electrophoresis (SDS-PAGE), and then tr- ansferred to PVDF membrane. After that, the protein transferred PVDF membrane was probed with primary antibodies of caspase-3, AKT, p-AKT, eNOS, p-eNOS, and GAPDH respec-tively. After that, the PVDF membrane was incu-bated with HPR-conjugated secondary antibod-ies. Finally, target protein bands were visual-ized by chemiluminescence, and GAPDH was used as the internal reference.

Statistical analysis

Data are presented as mean ± standard devia-tions (SD). Statistical comparisons were made by one-way analysis of variance (ANOVA) using SPSS software (version 18.0, USA), followed by Dunnet t multiple comparison test. P<0.05 was set as the significance level.

Results

Effects of XYJY on the cell viability of H2O2 in-duced PC12 cells

As can be seen from the Figure 1, cell viability was evaluated by CCK-8 assay. The results show that the cell viability was significantly reduced by treatment with H2O2 at the time points designed, including 24, 48, and 72 hours (p<0.01), compared to the normal cells. Furthermore, the results also indicate that XYJY can increase the cell viability of H2O2 induced PC12 cells from the concentration of 50 μg/ml compared to the control cells (p<0.01), and show a significant dose-dependent manner du-

cells (Figure 2). After exposure to H2O2, the per-centage of apoptotic cells sharply increased compared to the normal PC12 cells (p<0.01). However, our results also reveal that XYJY (100, 200 and 400 μg/ml) could dose-dependently reduce the percentage of apoptotic cells in H2O2 induced PC12 cells (p<0.01), compared to the control PC12 cells.

Effects of XYJY on ROS levels of H2O2 induced PC12 cells

As shown in Figure 3, flow cytometry assay with DCFH-DA staining was carried out to evaluate the effects of XYJY on ROS levels of H2O2 induced PC12 cells. After exposure to H2O2, the ROS levels of PC12 cells sharply increased compared to the normal PC12 cells (p<0.01). However, the present results also suggest that XYJY (100, 200 and 400 μg/ml) could dose-dependently reduce the ROS levels of PC12 cells induced by H2O2 (p<0.01), compared to the control PC12 cells.

Effects of XYJY on MDA, GSH and SOD of H2O2 induced PC12 cells

Furthermore, our present study also investigat-ed the effects of XYJY on levels of MDA, GSH and SOD in H2O2 induced PC12 cells (Figure 4). After exposure to H2O2, the levels of GSH-PX, and SOD were significantly decreased (p<0.01) whereas the MDA level was increased signifi-cantly (p<0.01), compared to the normal PC12 cells. However, the results show that XYJY treat-ments (100, 200 and 400 μg/ml) could signifi-

Figure 1. Protective effects of XYJY on H2O2 induced PC12 cells. The H2O2 con-centration used in the control and XYJY groups was 500 μmol/l, and CCK-8 assays were used to determine the cell viabilities of PC12 cells. Data were ex-pressed as mean ± SD (n=3), ##p<0.01, vs. normal; **p<0.01, vs. control.

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cantly reduce the levels of MDA (p<0.01) wh- ereas increase the levels of SOD and GSH-PX in H2O2 induced PC12 cells (p<0.01), compared to the control PC12 cells.

Effects of XYJY on caspase-3, P-AKT and P-eNOS in H2O2 induced PC12 cells

To explore the further molecular mechanisms for the activities of XYJY, the protein expression

of caspase-3, AKT, eNOS, p-AKT and p-eNOS in H2O2 induced PC12 cells were carried out (Figures 5 and 6). As shown in Figure 5, after treatment with H2O2, caspase-3 expression was significantly up-regulated compared to the normal group (p<0.01), and the XYJY treat-ments (200 and 400 μg/ml) significantly de- creased caspase-3 expression in H2O2 indu- ced PC12 cells (p<0.01), compared to normal cells.

Figure 2. Effects of XYJY on H2O2 induced apoptosis of PC12 cells. Data are expressed as mean ± SD (n=3), ##p<0.01, vs. normal; **p<0.01, vs. control.

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In addition, the results in Figure 6 also indicate that after treatment with H2O2, no obvious dif-ference could be found in the expression of AKT, eNOS, p-AKT and p-eNOS of PC12 cells (p>0.05), compared to the normal cells. How- ever, XYJY treatments (200 and 400 μg/ml) could significantly up-regulate expression of p-AKT (p<0.01) and p-eNOS (p<0.01) in H2O2 induced PC12 cells, compared to the control cells.

Discussion

The present study shows that Xiaoyao-jieyu-san (XYJY), an empirical TCM prescription for treatment of stroke and its related diseases in our hospital, possesses promising protec- tive effect on the oxidative damage induced by hydrogen peroxide (H2O2) in PC12 cells and its potential pharmacological mechanis- ms.

Figure 3. Effects of XYJY on ROS levels of H2O2 induced PC12 cells. Data are expressed as mean ± SD (n=3), ##p<0.01, vs. normal; **p<0.01, vs. control.

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Oxidative stress induced damage is one of the important molecular possible mechanisms for the tissue injury after ischemic stroke [10, 11]. In addition, reactive oxygen species (ROS) could result in oxidative damage of the bio-macromol-ecules and tissues of body, and subsequently lead to the lipid peroxidation, breakage of DNA and polypeptide, etc, and eventually leading to neuronal degeneration and necrosis [12, 13]. Hydrogen peroxide (H2O2) is a commonly used reagent for preparation of oxidative damaged

cells due to the H2O2 could easily go through the cytoplasmic membrane and form the pow-erful radicals (such as hydroxyl radical and sin-glet oxygen) via Fenton reaction with transition metal [14]. In addition, H2O2 could also induce the release of cytochrome C from the mitochon-dria, and subsequently result in the activation of caspase-3 which is a crucial activated death protease and bio-marker for cells undergoing apoptosis, leading to the apoptosis of cells [13, 15]. In the present study, H2O2 induced oxida-

Figure 4. Effects of XYJY on MDA, GSH, and SOD of H2O2 induced PC12 cells. Data are expressed as mean ± SD (n=3), ##p<0.01, vs. normal; **p<0.01, vs. control.

Figure 5. Effects of XYJY on protein expression of caspase-3 in H2O2 induced PC12 cells. Data are expressed as mean ± SD (n=3), ##p<0.01, vs. normal; **p<0.01, vs. control.

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tive damaged PC12 cells were successfully pre-pared to investigate the related activities of XYJY, and the results showed that XYJY could significantly alleviate the cell viability inhibition and apoptosis induced by H2O2 in PC12 cells. Furthermore, intracellular MDA, SOD, and GSH are important biomarkers to evaluate the oxida-tive stress. MDA is the production of peroxida-tion of membrane lipids induced by ROS, which could result in membrane damage and destruc-tion; while SOD and GSH are important anti-oxidant enzymes in mammalian cells [16, 17]. In the present study, XYJY could significantly reduce MDA whereas it could increase SOD and GSH in H2O2 induced PC12 cells. In addition, the results of the flow cytometry assay with DCFH-DA staining also indicated the XYJY could obviously decrease the ROS levels in H2O2 induced PC12 cells. All these results suggest that XYJY could significantly reduce the oxida-tive damage of H2O2 induced PC12 cells. AKT plays important role in the growth, proliferation, migration of nerve cells, and previous research-es have revealed that activation (phosphoryla-tion) of the AKT could protect the nerve cells from cell apoptosis induced by oxidative toxins, consequently it has been reported that activa-tion of AKT pathways might potentially be a new

target for treating ischemic stroke [18, 19]. Furthermore, NO mediated by eNOS could pro-tect the nerve cell and tissues, and up-regula-tion of eNOS would be beneficial for treatment of ischemic stroke [20, 21]. In summary, these data show that XYJY treatment could signifi-cantly up-regulation of P-AKT and P-eNOS in H2O2 induced PC12 cells.

Conclusion

In conclusion, the results presented here sug-gest that XYJY has protective effect against oxi-dative damage in PC12 cells mediated by regu-lation of AKT/eNOS signaling pathway. The present study would be beneficial for interpre-tation of the possible molecular mechanisms corresponding to the curative effects of XYJY against stroke and its related diseases.

Acknowledgements

This study was supported by the Shanghai Natural Science Foundation (grant no. 16ZR- 1433700).

Disclosure of conflict of interest

None.

Figure 6. Effects of XYJY on protein expression of p-AKT and p-eNOS in H2O2 induced PC12 cells. Data are expressed as mean ± SD (n=3), ##p<0.01, vs. normal; **p<0.01, vs. control.

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Address correspondence to: Dr, Bangjiang Fang, Department of Emergency, Longhua Hospital Sh- anghai University of Traditional Chinese Medicine, 725 Wanping Road, Xuhui District, Shanghai 200032, China. Tel: +86-021-64385700; E-mail: 15618075507@163.com

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