RESEARCH ARTICLE Open Access

Chinese medicine Di-Huang-Yi-Zhi protectsPC12 cells from H2O2-induced apoptosis byregulating ROS-ASK1-JNK/p38 MAPKsignalingLi-Min Zhang1, Rong-Rong Zhen1, Chao Gu1, Tian-Li Zhang1, Yue Li1, Miao Jin1, Bing Hu2* and Hong-Mei An3*

Abstract

Background: Oxidative stress mediates the nerve injury during the pathogenesis of Alzheimer’s disease (AD).Protecting against oxidative stress damage is an important strategy to prevent and treat AD. Di-Huang-Yi-Zhi(DHYZ) is a Chinese medicine used for the treatment of AD, but its mechanism remains unknown. This study isaimed to investigate the effect of DHYZ on H2O2 induced oxidative damage in PC12 cells.

Methods: PC12 cells were treated with H2O2 and DHYZ. Cell proliferation was detected by Cell counting kit-8(CCK-8) assay. Cytotoxicity of H2O2 was measured by lactate dehydrogenase (LDH) release assay. Apoptosiswere identified by Annexin V-FITC/PI staining. Caspase 3 activity was detected by commercial kit. Mitochondrial membranepotential (MMP) was detected by JC-1 staining. Reactive oxygen species (ROS) was 2′, 7′-Dichlorodihydrofluoresceindiacetate (DCFH-DA) staining. Protein expression and phosphorylation was identified by western blot.

Results: The results showed that DHYZ antagonized H2O2-mediated cytotoxicity and proliferation inhibition. DHYZ reducedROS production, stabilize mitochondrial membrane potential, inhibit Caspase-3 activity and apoptosis induced by H2O2. Inaddition, DHYZ inhibited the phosphorylation of ASK1, JNK1/2/3 and p38 MAPK which were up-regulated by H2O2.

Conclusions: The present study suggested that DHYZ protected PC12 cells from H2O2-induced oxidative stress damageand was related to inhibition of ROS production and ASK1-JNK/p38 MAPK signaling. The present study providesexperimental evidence for the application of DHYZ for the management of oxidative stress damage and AD.

Keywords: Alzheimer’s disease, Oxidative stress, Chinese herb, Di-Huang-Yi-Zhi, PC12 cells, Apoptosis, Signal transduction

BackgroundAlzheimer’s disease (AD) is an age-related degenerativedisease of the central nervous system. AD presents asprogressive cognitive impairment, and is closely relatedto β-amyloid (Aβ) and Tau pathology [1–3]. Both Aβand Tau can cause oxidative stress (OS) [4–6]. OS canmediate nerve injury and participate in the pathogenesisof AD [7, 8]. Reactive oxygen species (ROS) are the main

effectors in the OS process, and can oxidize proteins,lipids and DNA, affect mitochondrial function, activateCaspase-3, promote neuronal apoptosis, and thus par-ticipate in the pathogenesis of AD [9, 10]. Interventionof OS damage is an important strategy for the preven-tion and treatment of AD [11, 12].Traditional Chinese medicine (TCM) is known to play

an important role in the prevention and treatment ofAD. Based on the theory of TCM, clinical medicationand related studies, we established a Chinese herbalformula Di-Huang-Yi-Zhi (DHYZ). DHYZ consists ofShu-Di (prepared root of Rehmannia glutinosa (Gaert.)Libosch. ex Fisch. et Mey.), Yi-Zhi-Ren (fruits of Alpiniaoxyphylla Miq.), Shi-Chang-Pu (root of Acorus tatari-nowii Schott), Fu-Shen (Poria with hostwood) and

© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: beearhu@hotmail.com; beearhu@shutcm.edu.cn;lhsoatp@163.com2Department of Oncology, Institute of Traditional Chinese Medicine inOncology, Longhua Hospital, Shanghai University of Traditional ChineseMedicine, Shanghai 200032, China3Department of Science & Technology, Longhua Hospital, ShanghaiUniversity of Traditional Chinese Medicine, Shanghai 200032, ChinaFull list of author information is available at the end of the article

BMC ComplementaryMedicine and Therapies

Zhang et al. BMC Complementary Medicine and Therapies (2020) 20:54 https://doi.org/10.1186/s12906-020-2834-8

http://crossmark.crossref.org/dialog/?doi=10.1186/s12906-020-2834-8&domain=pdfhttp://orcid.org/0000-0003-0810-6670http://creativecommons.org/licenses/by/4.0/http://creativecommons.org/publicdomain/zero/1.0/mailto:beearhu@hotmail.commailto:beearhu@shutcm.edu.cnmailto:lhsoatp@163.com

Dan-Shen (root of Salvia miltiorrhiza Bunge) (Chinesepatent ZL2008102047153.3). All these herbs are effectivefor the prevention and treatment of AD.Previous studies have shown that DHYZ can antagonize

Aβ-mediated neurotoxicity and inhibit Aβ-induced neuro-cyte apoptosis in vitro [13]. DHYZ can also reduce synap-tic loss, antagonize Aβ-mediated nerve injury and inhibitphosphorylation of Tau protein, thereby improving thelearning and memory abilities of AD mice and rats[14, 15]. DHYZ can enhance the therapeutic effect ofDonepezil on AD and Parkinson’s disease dementia,improve clinical symptoms, cognitive ability and dailylives of patients [16, 17]. The effect of DHYZ on OSdamage remains unknown. The present study ex-plored the protective effect of DHYZ on OS mediatedby H2O2 in PC12 cells.

Materials and methodsChemicals and reagentsRoswell Park Memorial Institute (RPMI)-1640 mediumwas purchased from Corning (Manassas, VA). Fetal bo-vine serum (FBS), penicillin, streptomycin and trypsinwere purchased from Gibco (Grand Island, NY). Caspase3 activity assay kit, cell counting kit-8 (CCK-8), lactatedehydrogenase (LDH) cytotoxicity assay kit, mitochondrialmembrane potential (MMP) assay kit, N-acetyl-L-cysteine(NAC), and reactive oxygen species (ROS) assay kit werepurchased from Beyotime Biotechnology (Haimen, Jiangshu,China). Apoptosis detection kit was from BD Biosciences(San Jose, CA, USA). Antibodies against Apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase(JNK)1/2/3 were purchased from Abcam (Cambridge, UK).Antibodies against glyceraldehyde-3-phosphate dehydrogen-ase (GAPDH), p-ASK1 (S83), and p-JNK1/2/3 (T183/Y185)were from Bioworld (St. Louis Park, MN). Antibodiesagainst p38 mitogen-activated protein kinase (MAPK) andp-p38 MAPK (T180/Y182) were purchased from CellSignaling Technology (Danvers, MA).

DHYZ extractionHerbs in DHYZ are Shu-Di (prepared root of R. gluti-nosa (Gaert.) Libosch. ex Fisch. et Mey.), Yi-Zhi-Ren(fruits of A. oxyphylla Miq.), Shi-Chang-Pu (root of A.tatarinowii Schott), Fu-Shen (Poria with hostwood) andDan-Shen (root of S. miltiorrhiza Bunge) (Chinese pa-tent ZL2008102047153.3). All herbs were obtained fromLonghua Hospital and identified by Professor Liwen Xufrom Shanghai University of Traditional Chinese Medi-cine, Shanghai, China. Voucher specimen is deposited inInstitute of Traditional Chinese Medicine in Oncology,Longhua Hospital, Shanghai University of TraditionalChinese Medicine, Shanghai, China (specimen number:DHYZ-001). Extraction and quality control of DHYZhas been previously described, Salvianolic acid B was

used as a reference phytochemical [13, 18–20]. DHYZextract was dissolved in serum-free RPMI-1640 medium,passed through 0.22 μm filter for sterilization and storedat − 20 °C.

Cell culturePC12 cells were purchased from The Cell Bank of TypeCulture Collection of Chinese Academy of Sciences.PC12 cells were cultured in RPMI-1640 containing 10%FBS, 100 U/mL penicillin and 100 mg/mL streptomycin,and maintained at 37 °C in a humidified incubator with5% CO2. PC12 cells in logarithmic growth phase wereused for subsequent experiments.

Cell proliferation assayPC12 cells were seeded in a 96-well plate (1 × 104/well).After 24 h of culture, PC12 cells were treated with differ-ent concentrations of DHYZ or the same volume ofserum-free RPMI-1640 for 24 h, or 400 μM of H2O2 for4 h. CCK-8 reagents were used to detect cell prolifera-tion according to the manufacturer’s instructions. Cellsurvival rate was calculated by the following formula:Cell survival (%) = (experimental OD value/control ODvalue) × 100%.

LDH cytotoxicity assayPC12 cells were plated in a 96-well plate (1 × 104/well).After 24 h of culture, DHYZ (50–200 μg/mL) or thesame volume of serum-free RPMI-1640 were added.After 24 h of treatment, the cells were treated with H2O2(400 μM) for 4 h. The amount of LDH released in eachgroup was measured according to the manufacturer’sinstructions. The results were expressed as fold of non-treated normal group.

Detection of apoptosisPC12 cells treated with DHYZ and H2O2 were collectedand washed with PBS. The cells were suspended in100 μl Binding Buffer. Annexin V-FITC and propidiumiodide (PI) (5 μl each) were added, mixed and incubatedfor 15 min at room temperature, and then 400 μl BindingBuffer was added and mixed. Cell apoptosis was detectedby flow cytometry (BD Biosciences, San Jose, CA).

MMP detectionPC12 cells were incubated in a 24-well plate (5 × 104/well). After 24 h of culture, different concentrations ofDHYZ or the same volume of serum-free RPMI-1640were added. After 24 h, PC12 cells were treated with400 μM H2O2 for 4 h. JC-1 staining was performed ac-cording to the manufacturer’s instructions. Fluorescencemicroscopy and flow cytometry were used to identifypositive JC-1 staining.

Zhang et al. BMC Complementary Medicine and Therapies (2020) 20:54 Page 2 of 8

Detection of Caspase-3 activityPC12 cells treated with DHYZ and H2O2 were collected,and the activity of Caspase-3 was detected according tothe manufacturer’s manual.

ROS detectionThe ROS production was detected according to themanufacturer’s instructions. Briefly, PC12 cells were cul-tured in a 6-well plate (2.5 × 105 cells/well). After 24 h ofculture, DHYZ (50–200 μg/mL) or the same volume ofserum-free RPMI-1640 were added. After 24 h, the cellswere treated with 400 μM of H2O2 for 4 h. For ROS de-tection, the cells were incubated with 2′, 7′-Dichlorodi-hydrofluorescein diacetate (DCFH-DA) (10 μmol/L) for30 min at 37 °C, observed under a fluorescence micro-scope and quantified with a fluorescence microplatereader (Thermo Fischer Scientific, Waltham, MA). ForROS inhibition, DHYZ-treated PC12 cells were incu-bated with NAC (500 μM) for 2 h followed by H2O2treatment.

Western blotPC12 cells treated with DHYZ and H2O2 were collected,lysed in RIPA buffer and quantified using the BCA kit.Proteins were separated by 8–10% SDS-PAGE electro-phoresis and transferred to PVDF membrane on a semi-dry transfer unit. The membranes were blocked with 5%non-fat milk for 2 h, incubated with antibodies againstASK1, p-ASK1, JNK1/2/3, p-JNK1/2/3, p38 MAPK andp-p38 MAPK (1:800) or GAPDH (1:2000) at 4 °C over-night. The blots were washed with TBST and incubatedwith secondary antibody (1:5000) at 37 °C for 2 h. Thebands were visualized by the ECL method. Proteins ex-pression were quantified by Image J software.

Statistical analysisAll data were analyzed by SPSS 21.0 software, and theresults were expressed as mean ± standard deviation(SD). Intergroup differences were analyzed by one-wayanalysis of variance (ANOVA) and LSD-t or Dunnett’stest. P < 0.05 was considered as significant difference.

ResultsDHYZ attenuates H2O2-mediated cytotoxicityWe first observed the effects of H2O2 and DHYZ onPC12 cell proliferation. As shown in Fig. 1, 100–600 μMof H2O2 significantly inhibited the proliferation of PC12cells (P < 0.01), and the 50% inhibitory concentration(IC50) was about 400 μM. Low doses of DHYZ (25–400 μg/mL) showed no significant effect on the prolifera-tion of PC12 cells. Based on these observations, 400 μMof H2O2 and 50–200 μg/mL of DHYZ were selected forsubsequent experiments. Further study revealed that

DHYZ could antagonize the inhibitory effects of H2O2on proliferation of PC12 cells (P < 0.05).The cytotoxicity of H2O2 on PC12 cells was detected

by the LDH release assay. LDH is released from cellswhen cell membrane is damaged, and thus can reflectcell damage and cytotoxicity. The results showed thatthe LDH release from PC12 cells increased after H2O2treatment (P < 0.01). After DHYZ treatment, the H2O2-induced LDH release was reduced (P < 0.05). Theseresults suggested that DHYZ could antagonize H2O2-mediated cytotoxicity.

DHYZ antagonizes H2O2-induced apoptosisApoptosis contributes to oxidative stress-mediated neur-onal damage [21]. In the present study, Annexin V-FITCand PI double staining were used to detect apoptosis byflow cytometry. The results showed that H2O2 couldpromote apoptosis in PC12 cells (P < 0.01). Apoptosis ofPC12 cells was significantly decreased after DHYZ treat-ment in a dose-dependent manner (P < 0.01) (Fig. 2).

DHYZ undermines H2O2-induced reduction of MMPMitochondria are important organelles that regulateapoptosis. In this study, MMP was detected by JC-1staining. JC-1 accumulates in the mitochondrial matrix,forms J-aggregates and produces red fluorescence whenthe MMP is high, while JC-1 exists as a monomer andemits green fluorescence when the MMP is low. ThePC12 cells showed green fluorescence after H2O2 treat-ment. Upon DHYZ treatment, the red fluorescence inten-sity of PC12 cells increased and the green fluorescencedecreased (Fig. 3) (P < 0.05). These results suggested thatDHYZ could reverse H2O2-induced reduction of MMP.

DHYZ inhibits H2O2-activated Caspase-3Caspase-3 is an executive protease in the apoptoticprocess, which is regulated by mitochondria and deathreceptor pathway [22]. In this study, a specific enzymesubstrate was used to detect the activity of Caspase-3.The results showed that the activity of Caspase-3 in-creased significantly in PC12 cells after H2O2 treatment(P < 0.01). DHYZ inhibited H2O2-activated Caspase-3 ina dose-dependent manner (Fig. 4) (P < 0.05).

Effect of DHYZ on H2O2-induced ROS productionH2O2 can produce HO• to damage cells [23]. We observedROS production by DCFH-DA staining. DCFH-DA enterscells and generates DCFH under the action of esterases.DCFH generates fluorescent DCF under the action of ROS.The fluorescence intensity of DCF can reflect ROS level.The results showed that the green fluorescence of PC12cells was enhanced after H2O2 treatment (P < 0.01), butDHYZ treatment decreased the green fluorescence in adose-dependent manner (P < 0.05) (Fig. 5A and B)

Zhang et al. BMC Complementary Medicine and Therapies (2020) 20:54 Page 3 of 8

suggested that DHYZ could reduce H2O2-inducedROS production.NAC was further used to block the ROS production.

The results showed that 500 μM of NAC significantlyinhibited ROS production, and also antagonized the

effects of H2O2 on Caspase-3 activity and apoptosis (P <0.01). NAC also attenuated the effects of DHYZ onCaspase-3 activity and apoptosis (P < 0.05) (Fig. 5C-F).These observations suggested that ROS participated inthe effects of H2O2 and DHYZ.

Fig. 1 Effects of DHYZ on H2O2-mediated cytotoxicity. PC12 cells were treated with H2O2 for 4 h (a) or DHYZ for 24 h (b) and cell proliferationwas detected by CCK-8 assay. PC12 cells were pre-treated with DHYZ for 24 h, followed by H2O2 treatment for 4 h and subjected to CCK-8 assay(c) and LDH release assay (d). △P < 0.01, versus normal group; *P < 0.05, **P < 0.01, ***P < 0.001, versus control group. The data are mean ± SD ofthree independent experiment each in triplicate

Fig. 2 Effects of DHYZ on H2O2-induced apoptosis. DHYZ and H2O2 treated PC12 cells were stained with Annexin V/PI, apoptosis was identifiedby flow cytometry (A) and expressed as mean ± standard deviation (the sum of early apoptosis (right lower quadrant) and late apoptosis (rightupper quadrant)) (B). a, normal group; b, H2O2 group; c, control group; d, H2O2 + DHYZ (50 μg/ml) group; e, H2O2 + DHYZ (100 μg/ml) group; f,H2O2 + DHYZ (200 μg/ml) group.

△P < 0.01, versus normal group; ***P < 0.001, versus control group. The data are mean ± SD of three independentexperiment each in triplicate

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Effect of DHYZ on H2O2 activated ASK1-JNK/p38 MAPKsignalingROS can activate ASK1-JNK/p38 MAPK signal trans-duction [24, 25]. We detected the expression and phos-phorylation of ASK1-JNK/p38 MAPK by Western blot.As shown in Fig. 6, H2O2 up-regulated the phosphoryl-ation of ASK1, JNK and p38 MAPK, without affectingtheir expressions. DHYZ inhibited the phosphorylationof ASK1, JNK and p38 MAPK induced by H2O2.

DiscussionBrain, as an organ with high oxygen consumption, isprone to accumulate ROS during the metabolic process.

In addition, fewer antioxidant enzymes can penetrate theblood-brain barrier, and the brain contains more unsat-urated fatty acids and transition metals, which also leadto ROS accumulation, promote synapse and neuronalloss, and accelerate the progress of AD [11, 26] Anti-oxidative damage and maintenance of normal neuronalfunction are important strategies to prevent and treatAD [12, 26]. The present study showed that H2O2 hadcytotoxic effect on PC12 cells and inhibited cell prolifer-ation. DHYZ could antagonize the cytotoxicity and pro-liferation inhibition of H2O2, suggesting that DHYZ hadneuroprotective effect.Under physiological condition, the body is in an equi-

librium state of redox. During oxidative stress, excessiveROS accumulation in cells lead to irreversible mitochon-drial permeability, transition pore opening, decreasedMMP, release of cytochrome C from mitochondria, suc-cessive activation of Caspase-9 and Caspase-3, and apop-tosis initiation [27–29]. The present study showed thatapoptosis of PC12 cells was observed after H2O2 treat-ment, accompanied by decrease of MMP and increase ofCaspase-3 activity, suggesting that H2O2 could induceapoptosis through the mitochondrial pathway. UponDHYZ treatment, the effect of H2O2 on apoptosis wasantagonized.ROS is an effector of oxidative stress in cells. The

present study showed that H2O2 increased the ROS levelin PC12 cells. NAC-mediated inhibition of ROS couldantagonize the effects of H2O2 on apoptosis andCaspase-3, suggesting that H2O2 mediated oxidative

Fig. 3 Effects of DHYZ on H2O2 down-regulated MMP. DHYZ and H2O2 treated PC12 cells were stained with JC-1 and observed under a fluorescencemicroscope (× 200) (A), detected by flow cytometry (B) and JC-1 fluorescence were expressed as 100% of total (C). a, normal group; b, H2O2 group; c,control group; d, H2O2 + DHYZ (50 μg/ml) group; e, H2O2 + DHYZ (100 μg/ml) group; f, H2O2 + DHYZ (200 μg/ml) group. △P < 0.01, versus normalgroup; *P < 0.05, **P < 0.01, ***P < 0.001, versus control group. The data are mean ± SD of three independent experiment each in triplicate

Fig. 4 Effects of DHYZ on H2O2-activated Caspase-3. DHYZ and H2O2treated PC12 cells were subjected to Caspase-3 activity detection.△P < 0.01, versus normal group; *P < 0.05, **P < 0.01, ***P < 0.001,versus control group. The data are mean ± SD of three independentexperiment each in triplicate

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Fig. 5 Effects of DHYZ on H2O2-induced ROS production. DHYZ and H2O2 treated PC12 cells were stained with DCFH-DA, observed under afluorescence microscope (× 200) (A), detected by fluorescence microplate reader and expressed as fold of non-treated normal group (B). DHYZtreated PC12 cells were incubated with NAC for 2 h, followed by H2O2 treatment and subjected to ROS (C), Caspase-3 activity (D), apoptosisdetection (E) and expressed as 100% of total (F). a, normal group; b, H2O2 group; c, control group; d, H2O2 + DHYZ (50 μg/ml) group; e, H2O2 +DHYZ (100 μg/ml) group; f, H2O2 + DHYZ (200 μg/ml) group.

△P < 0.01, versus normal group; *P < 0.05, **P < 0.01, ***P < 0.001, versus control groupor versus NAC (−) group. The data are mean ± SD of three independent experiment each in triplicate

Fig. 6 Effects of DHYZ on H2O2-activated proteins phosphorylation. DHYZ and H2O2 treated PC12 cells were subjected to western blot withindicated antibodies (a, c and e) and quantified by Image J software (b, d and F). △P < 0.01, versus normal group; *P < 0.05, **P < 0.01, ***P < 0.001,versus control group. The data are mean ± SD of three independent experiment each in triplicate

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stress damage in PC12 cells through ROS. Moreover,NAC could also reduce the protective effect of DHYZon PC12 cells, suggesting that inhibiting ROS was an im-portant mechanism of DHYZ.ROS can activate ASK1-JNK/p38 MAPK signal transduc-

tion [24, 25, 30, 31]. ROS can promote ASK1 phosphoryl-ation, activate JNK or p38 MAPK, regulate downstreamapoptosis-related proteins, and induce neuronal apoptosis.Inhibiting JNK and MAPK can protect against nerve dam-age [32, 33]. The present study showed that phosphoryl-ation of ASK1, JNK and p38 MAPK were up-regulatedafter H2O2 treatment. DHYZ could also inhibit the phos-phorylation of ASK1, JNK and p38 MAPK. These obversionsuggested that ASK1, JNK and p38 MAPK participated inthe effect of DHYZ.DHYZ is established according to the TCM princi-

ples. Shu-Di (prepared root of R. glutinosa (Gaert.)Libosch. ex Fisch. et Mey.) is the Monarch (Jun) herband used for tonifying kidney. Shi-Chang-Pu (root ofA. tatarinowii Schott) and Dan-Shen (root of S.miltiorrhiza Bunge) are the Minister (Chen) herbsand used for dissolving stasis and phlegm, and calm-ing mental-state. Fu-Shen (Poria with hostwood) isthe Assistant (Zuo) herb and used for calming spirit.Yi-Zhi-Ren (fruits of A. oxyphylla Miq.) is the Guide(Shi) herb and used as herb for warming kidney. Allthose herbs are synergistically acted in DHYZ fromthe perspective of TCM and have showed beneficialfor AD treatment [13, 34–38].The main components of DHYZ including Verbasco-

side, Catalpinoside, Rehmannioside A, OxyphyllenodiolA and B, Stigmasterol, Apigenin, Cycloartenol, Tramete-nolic acid B, Asatone, Bissarcin, Tanshinone IIA and Sal-vianolic acid B [18–20, 36]. Verbascoside protects PC12cells from 1-methyl-4-phenylpyridinium ion (MPP(+))induced neurotoxicity via down-regulation of extracellularhydrogen peroxide level [39]. Other compounds, such asCatalpinoside, Rehmannioside A, Stigmasterol, Apigenin,Trametenolic acid B, Asatone, Tanshinone IIA and Salvia-nolic acid B have showed neuro-protective effect againstoxidative stress damage through different mechanism[40–47]. However, the role of those compounds in DHYZneed further explore.

ConclusionsIn summary, the present study suggested that DHYZprotected PC12 cells from H2O2-induced oxidativestress damage, and its mechanism was related toinhibition of ROS production and ASK1-JNK/p38MAPK phosphorylation. The present study providedexperimental evidence for alleviating oxidative stressdamage, preventing and treating AD and neurode-generative diseases by TCM.

AbbreviationsAD: Alzheimer’s disease; ANOVA: Analysis of variance; ASK1: Apoptosis signal-regulating kinase 1; Aβ: β-amyloid; CCK-8: Cell counting kit-8; DCFH-DA: 2′,7′-Dichlorodihydrofluorescein diacetate; DHYZ: Di-Huang-Yi-Zhi;GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; IC50: 50% inhibitoryconcentration; JNK: c-Jun N-terminal kinase; LDH: Lactate dehydrogenase;MMP: Mitochondrial membrane potential; NAC: N-acetyl-L-cysteine;OD: Optical density; OS: Oxidative stress; p38 MAPK: p38 mitogen-activatedprotein kinase; PI: Propidium iodide; ROS: Reactive oxygen species; RPMI-1640: Roswell Park Memorial Institute – 1640; SD: Standard deviation; SDS-PAGE: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis;TCM: Traditional Chinese medicine

AcknowledgementsNot applicable.

Authors’ contributionsHMA established the herbal formula; HMA and BH designed the study,coordinated technical support and funding; HB revised the manuscript; LMZperformed the study and drafted the manuscript; RRZ, CG, TLZ, YL and MJparticipated the study. All authors read and approved the final manuscript.

FundingThis study was supported by National Natural Science Foundation of China(grant number 81873246), Natural Science Foundation of ShanghaiMunicipality (grant number 18ZR1439900), and Scientific and TechnologicalInnovation Projects of Longhua Hospital (grant number CX201766).

Availability of data and materialsThe datasets used and/or analyzed during the current study available fromthe corresponding author on reasonable request.

Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Neurology, Longhua Hospital, Shanghai University ofTraditional Chinese Medicine, Shanghai 200032, China. 2Department ofOncology, Institute of Traditional Chinese Medicine in Oncology, LonghuaHospital, Shanghai University of Traditional Chinese Medicine, Shanghai200032, China. 3Department of Science & Technology, Longhua Hospital,Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China.

Received: 15 November 2019 Accepted: 27 January 2020

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Zhang et al. BMC Complementary Medicine and Therapies (2020) 20:54 Page 8 of 8

AbstractBackgroundMethodsResultsConclusions

BackgroundMaterials and methodsChemicals and reagentsDHYZ extractionCell cultureCell proliferation assayLDH cytotoxicity assayDetection of apoptosisMMP detectionDetection of Caspase-3 activityROS detectionWestern blotStatistical analysis

ResultsDHYZ attenuates H2O2-mediated cytotoxicityDHYZ antagonizes H2O2-induced apoptosisDHYZ undermines H2O2-induced reduction of MMPDHYZ inhibits H2O2-activated Caspase-3Effect of DHYZ on H2O2-induced ROS productionEffect of DHYZ on H2O2 activated ASK1-JNK/p38 MAPK signaling

DiscussionConclusionsAbbreviationsAcknowledgementsAuthors’ contributionsFundingAvailability of data and materialsEthics approval and consent to participateConsent for publicationCompeting interestsAuthor detailsReferencesPublisher’s Note