Sage Journals: Discover world-class research

Abstract

As the aging phenomenon continues to increase, the incidence of neurodegenerative diseases continues to increase annually. As one of the significant contributive factors of neurodegenerative diseases, oxidative stress damage has received extensive attention in recent years. Oxidative stress plays an important role in neuronal damage through various apoptotic mechanisms related to neurodegenerative diseases. The use of natural antioxidants to combat oxidative stress may be a useful approach in delaying disease progression. In this study, we explored the neuroprotective effect of hyperoside on rat pheochromoma (PC12) cells. Specifically, the antioxidant effect and mechanism of hyperoside in hydrogen peroxide (H₂O₂)-induced cellular cytotoxicity were investigated. Our results showed that hyperoside could significantly increase the survival rate of rat PC12 cells when exposed to H₂O₂. In addition, hyperoside regulated the expression of genes and proteins in the corresponding pathways by up-regulating the phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt), and light chain 3β (LC3B) pathways and down-regulating the nuclear factor-ᴋ-gene binding (NF-κB), Bcl2-associated X (Bax), cysteinyl aspartate specific proteinase 3 (Caspase 3), and P62 pathways, thereby inhibiting cell apoptosis. Therefore, hyperoside can effectively inhibit H₂O₂-induced oxidative stress damage by regulating inflammation, autophagy, and apoptosis-related pathways.

Keywords

neurodegenerative diseases hyperoside oxidative stress F-ᴋB

Neurodegenerative diseases are caused by progressive neuronal death in the brain and spinal cord, leading to damage to the nervous system resulting in cognitive and motor dysfunction.^1,2 Several studies have shown that oxidative stress is closely related to the pathogenesis of neurodegenerative diseases.^3
-5In vivo studies show that the imbalance of oxidative and antioxidant systems is the cause of oxidative stress. Oxidative stress produces a large number of free radicals. Neurons are very sensitive to free radicals and can be easily damaged. The causes of oxidative stress also include protein misfolding caused by excessive reactive oxygen species (ROS), activation of glial cells, and mitochondrial dysfunction leading to apoptosis.^6
-8 Therefore, the improvement of or reduction in oxidative stress injury is closely related to the management of neurodegenerative diseases.

Hyperoside {quercetin 3-O-β-D-galactoside (Figure 1)}, is a flavanol glycoside found in Acanthopanax senticosus (Rupr. & Maxim.) Harms (Araliaceae) .⁹ This compound is effective in alleviating oxidative injury, inhibiting apoptosis, and reducing inflammation.¹⁰ Studies report that hyperoside can protect rat cardiomyocytes from apoptosis induced by ischemia-reperfusion injury.¹¹ It has also been shown that hyperoside can reduce H₂O₂-induced osteoblast MC3T3-E1 phosphorylation Jun terminal kinase (JNK) and lower P38 levels by inhibiting mitogen-activated protein kinase (MAPK) signaling; it reduces H₂O₂-induced cell apoptosis and apoptosis-related proteins.¹² However, there are only a few studies that have explored the protective mechanism of hyperoside in alleviating the oxidative stress injury of neurons. In this study, PC12 rat pheochromocytoma cells, a sympathetic nervous system tumor-cell line widely used to study neuronal injury-related models and highly similar to dopaminergic neurons in structure and function, were selected to establish an H₂O₂-induced oxidative-injury model. Herein, we explored the mechanism of hyperoside in protecting PC12 cells from damage owing to oxidative stress.

Figure 1

Structure of hyperoside.

Materials and Methods

Reagents and Drugs

Hyperoside was purchased from Shanghai Yuanye Biological Technology Co., Ltd. Dulbecco’s modified Eagle medium (DMEM) and MTT were obtained from Beijing Solarbio Sciences & Technology Co., Ltd., fetal bovine serum (FBS) from Genimi Biological Products Co., Ltd, anti-phospho-PI3K P85 antibody (Y458)/p55/(Y199), anti-PI3K antibody (19H8), anti-phospho Akt antibody (S473) (D9E), anti-Akt antibody (ab179463)(EPR167), anti-phospho NF-κB P65 antibody (bs-17502R), and anti-NF-κB P65 antibody (bs-20160R) from Cell Signaling Technology Inc., anti-Bax antibody (50599, 2-lg), anti-caspase 3 antibody (bs-0081R), anti-LC3B-specific antibody (16396, 1-AP), and anti-P62 antibody (18420, 1-AP) from Proteintech, and β-actin (GB11001) from Servicebio.

Cell Culture and Modeling

PC12 cell line (Institute of Basic Medicine, Chinese Academy of Medical Sciences.) was cultured in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin, in an incubator with 5% CO₂ at 37 °C. PC12 cells were inoculated in a 96-well-plate at a density of 1.25 × 10⁴ cells/well for 24 hours. PC12 cells were treated with 10% H₂O₂ diluted to different modeling concentrations (700, 600, 500, 400, 300, 200, 100, and 0 μmol/L) with DMEM. After 12 hours, 5 mg/mL of MTT (Beijing Mengyimei Biological Technology Co., Ltd) solution was added to each well and allowed to react for 4 hours, after which the absorbance was detected at 490 nm. The corresponding survival rate was calculated and H₂O₂ at half lethal concentration was used for further experiments. We selected hyperoside concentrations of 50, 25, and 12.5 µmol/L to pretreat PC12 cells for 12 hours, followed by treatment with 400 µmol/L H₂O₂ for 12 hours.

Cell Viability Assays

An MTT assay was used to determine the relative viability of PC12 cells after pretreatment with different concentrations of hyperoside. Cells were seeded in a 96-well plate at a density of 1.25 × 10⁴ cells/well with complete medium. After pretreatment with diﬀerent concentrations of hyperoside (50, 25, and 12.5 μmol/L) for 12 hours based on groupings, PC12 cells were then exposed to H₂O₂ for 12 hours as described in Section 2.2. Then, 20 µL MTT solution was added (final concentration 5 mg/mL). Subsequently, the supernatant was removed and the formazan crystals were dissolved in dimethyl sulfoxide. The absorbance of the samples was measured at 490 nm.

Reverse Transcription PCR (RT-PCR)

Highly differentiated PC12 cells were seeded in 6 cm cell culture plates at a concentration of 5 × 10⁵ cells/mL. After 24 hours of seeding, cells in the treatment group were treated with 50 μmol/L hyperoside for 12 hours, while control cells were treated with DMEM. All groups, except the control, were treated with 400 μmol/L H₂O₂ for 12 hours and total RNA was isolated from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. HiFi-MMLV cDNA kit (Cwbio, Beijing, China) was used to determine the composition of cDNA conforming to the specification. Primer sequences employed in RT-Qpcr are listed in the following:PI3K(Forward: 5’-ACACGGGGGCATTCAAAGAT-3’,Reverse:5’-GTCGTTGTGCCTGTCACCTA-3’),Akt1(Forward:5’-AGCATGGAGTGTGTGGACAG-3’,Reverse:5’-TACAGATGATCCATGCGGGG-3’),β-actint(F:5’-ACTCTGTGTGGATTGGTGGC-3’,R:5’-ACTCTGTGTGGATTGGTGGC-3’),Bax(F:5’-GCCTCCTTTCCTACTTCGGG-3’,R:5’-TTCCCCGTTCCCCATTCATC-3’),Caspase3(F:5’-TCTACCGCACCCGGTTACTA-3’,R:5’-TCAAATTCCGTGGCCACCTT-3’),NF-ƘB(F:5’-GGAGATGGCCCACTGCTATC-3’,R:5’-GCACCTTGGGATGCGTTTTT-3’),LC3B(F:5’-TCCTGAACCCCAGCCATTTC-3’,R:5’-GGCATGGACCAGAGAAGTCC-3’),P62(F:5’-GCCGCCTAAATGGAGTCTGA-3’,R:5’-ACAGTGCGAAACTGACAATGG-3’). The expression levels of PI3K, Akt, NF-κB, Bax, Caspase 3, LC3B, and P62 were determined using an SYBR V R Green PCR Kit (Takara). RT-PCR was conducted using an Applied Biosystems 7500 real-time PCR instrument (ABI, Foster, CA, USA).

Western Blot (WB) Analysis

PC12 cells were seeded in 6 cm cell culture plates at a concentration of 5 × 10⁵ cells/mL. After 24 hours of seeding, cells were treated with 50 µM hyperoside for 12 hours in the treatment group, while control cells were treated with DMEM. Next, cells were treated with 400 μmol/L H₂O₂ for 12 hours except for the control group. The total soluble proteins were extracted from cultured PC12 cells using RIPA buﬀer (Beyotime Institute of Biotechnology, Nantong, China) containing the protease inhibitor, PMSF. Protein concentrations were determined using a Pierce BCA protein assay (Thermo Fisher Scientiﬁc, USA). Equal quantities of protein samples were separated using SDS-PAGE and then transferred to PVDF membranes (EMD Millipore, USA). Membranes were blocked with 5% skim milk for 3 hours. Subsequently, the proteins were incubated with the following corresponding primary antibodies overnight at 4 °C: anti-β-actin (1:1000, GB11001, Servicebio), P-PI3K [1:500, Y458, Cell Signaling Technology, Inc (CST)], PI3K [1:250, 19H8, Inc (CST)], Akt [1:2000, ab179463, Inc (CST)], P-Akt [1:1000, s473, Inc (CST)], NF-κB [1:1000, bs-20160R, Inc (CST)], P-NF-κB [1:1000, bs-17502R, Inc (CST)], Bax (1:1000, 50599‐2-lg, Proteintech), Caspase 3 (bs-0081R, Bioss), P62 (1:1000, 18420‐1-AP, Proteintech), and LC3B (1:1000, 16396‐1-AP, Proteintech). After washing with TBST, membranes were incubated with HRP-linked secondary antibody (Zsbio, Beijing, China; 1:2000) for 2 hours. Lastly, ECL ultra-sensitive luminescence solution and a Western blotting detection kit were used to visualize the protein bands. Images were captured using a BIO-RAD gel imaging system and the grayscale was measured using Image J software.

Statistical Analysis

All experiments were performed at least three times and the results are expressed as means ± standard deviation (‾X ± SD). One-way analysis of variance was used to examine the differences between treatment groups. All statistical analyses were performed using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA). A difference was considered statistically signiﬁcant at ^# P < 0.05 or *P < 0.05.

Results

Protective Effect of Hyperoside on PC12 Cell Viability After H₂O₂ Treatment

PC12 cells were treated with different doses of hyperoside (0, 400 μmol/L). It was found that hyperoside had no significant effects on cell viability at doses ranging from 0 to 50 μmol/L (Figure 2(A)). As seen in Figure 2(B), hydrogen peroxide significantly decreased the viability of PC12 cells at doses of 200, 300, 400, 500, 600, and 700 μmol/L. Considering that cell viability was inhibited by 50% at 400 μmol/L H₂O₂, this dose was used to induce the oxidative stress injury in PC12 cells. Therefore, we selected hyperoside concentrations of 50, 25, and 12.5 µmol/L to pretreat PC12 cells for 12 hours, followed by treatment with 400 µmol/L H₂O₂ for 12 hours. The cell viability was found to be significantly higher than that of the model group (Figure 2(C)).

Figure 2

Protective effect of hyperoside on PC12 cell viability after H₂O₂ treatment. (A) Effect of different concentrations of hyperoside on the survival rate of PC12. (B) Effect of different concentrations of H₂O₂ on the survival rate of PC12. (C) Effect of pretreatment with hyperoside on H₂O₂-induced PC12 on cell survival rate. Data represent the ‾X ± S, n = 4,^# P < 0.05 vs Control group, *P < 0.05 vs Model group.

Increased PI3K and Akt mRNA and Its Phosphorylated Protein Expression in Hyperoside-Treated Cells

As shown in Figure 3, compared to the control group, RT-PCR and WB analysis showed that the expression of PI3K, Akt mRNA, and related proteins in the model group (400 μmol/L H₂O₂) was significantly down-regulated. After 12 hours of treatment of cells with hyperoside (50 μmol/L), the mRNA expression of PI3K and Akt was significantly higher than that of the cells induced by H₂O₂ (Figure 3(A) and (B)). As mRNA expression increased, the protein expression levels of P-PI3K and P-Akt also increased significantly (Figure 3(C), (D) and (E)). Our results showed that hyperoside may regulate oxidative stress damage by regulating the PI3K/Akt signaling pathway and significantly promote the expression of PI3K and Akt genes and corresponding proteins after oxidative damage.

Figure 3

Increased PI3K and Akt mRNA and phosphorylated protein expression in hyperoside-treated cells. (A) PI3K/ (B) Akt gene expression. (C) P-PI3K/PI3K and P-Akt/Akt protein expression. (D) P-PI3K/PI3K and (E) P-Akt/Akt protein expression. The data represent the‾X ± S, n = 3,^# P < 0.05 vs Control group, *P < 0.05 vs Model group.

Changes in NF-κB/Bax/Caspase 3 mRNA and phosphorylated protein expression in hyperoside-treated cells

As shown in Figure 4, compared to the control group, RT-PCR and WB analysis showed that the expression of NF-κB, Bax, Caspase 3 mRNA, and related proteins in the model group (400 µmol/L H₂O₂) was significantly up-regulated. After the cells were treated with hyperoside (50 μmol/L) for 12 hours, the mRNA expressions of NF-κB, Bax, and Caspase 3 were significantly lower than in those treated with H₂O₂ alone (Figure 4(A), (B) and (C)). While mRNA expression decreased, the expression levels of NF-κB, Bax, and Caspase 3 proteins also decreased significantly (Figure 4(D), (E), (F) and (G)). These results indicate that hyperoside may regulate oxidative stress damage by regulating the NF-κB/Bax/Caspase 3 signaling pathway and significantly promote the expression of NF-κB/Bax/Caspase 3 genes and related proteins after oxidative damage, thereby reducing oxidative stress damage.

Figure 4

Effected NF-κB/Bax/Caspase 3 mRNA and phosphorylated protein expression in hyperoside-treated cells. (A) NF-κB/ (B) Bax/(C) Caspase 3 gene expression. (D) NF-κB/Bax/Caspase3 protein expression. (E) P-NF-κB/NF-κB protein expression. (F) Relative Bax and (G) Caspase 3 protein expression. Data represent the‾X ± S, n = 3, ^# P < 0.05 vs Control group, *P < 0.05 vs Model group.

Changes in P62/LC3B mRNA and Phosphorylated Protein Expression in Hyperoside-Treated Cells

As shown in Figure 5, compared to the control group, RT-PCR and WB analysis showed that the expression of P62 mRNA and related proteins in the model group (400 µmol/L H₂O₂) increased significantly, while that of LC3B mRNA and related proteins decreased significantly. After 12 hours of treatment with hyperoside (50 μmol/L), the mRNA expression of P62 was significantly lower than that of H₂O₂-induced cells (Figure 5(A)). While mRNA expression decreased, the expression level of P62 protein was also significantly decreased (Figure 5(C) and D); the mRNA expression of LC3B was significantly higher than that of H₂O₂-induced cells (Figure 5(B)). While mRNA expression increased, the expression level of P62 protein also increased significantly (Figure 5(C) and (E)). The results indicate that hyperoside may regulate oxidative stress damage by regulating the P62/LC3B signaling pathway, significantly promoting the expression of LC3B genes and corresponding proteins after oxidative damage, and reducing the expression of P62 genes and corresponding proteins.

Figure 5

Effected P62/LC3B mRNA and phosphorylated protein expression in hyperoside-treated cells. (A) P62 /(B) LC3B gene expression. (C) P62/LC3B protein expression. (D) Relative P62 and (E) LC3B protein expression. Data represent the‾X ± S, n = 3, ^# P < 0.05 vs Control group, *P < 0.05 vs Model group.

Discussion

In our previous studies, we found that hyperoside, isolated from Acanthopanax senticosus, has significant anti-oxidative stress damage. It has been reported to alleviate the effects of oxidative stress damage, to varying degrees, in the treatment of inflammatory apoptosis.^12,13 H₂O₂ acts as an inducer of oxidative stress in vitro and can induce apoptosis in many different cell types, but H₂O₂ can be inhibited by antioxidants.¹⁴ Therefore, this study explored the role of hyperoside as an antioxidant and studied its protective mechanism in H₂O₂-treated PC12 cells. An MTT assay was used to study the proliferation of PC12 cells treated with hyperoside and our findings revealed that this flavonoid could effectively reduce the oxidative stress damage induced by H₂O₂ in these cells, consistent with the results reported in the literature.¹⁵

The PI3K/Akt signaling pathway is mediated by enzyme-linked receptors. Phosphatidylinositol-3-kinase (PI3K) and its downstream molecule, serine/threonine protein kinase B (protein kinase, PKB, alias Akt), constitute this pathway. This pathway plays an important role in protecting against oxidative stress by suppressing cell apoptosis and facilitating cell survival.¹⁶ Under certain circumstances, it can effectively inhibit neuronal apoptosis by activating the PI3K/Akt signaling pathway. Studies have shown that deuterium-depleted water (DDW) can inhibit H₂O₂-induced apoptosis in PC12 cells, reduce the generation of ROS, and increase the activities of catalase (CAT), copper, zinc SOD, and SOD. DDW may protect differentiated PC12 cells from the oxidative stimulation induced by H₂O₂ through the PI3K/Akt signaling pathway.¹⁷ Studies have also shown that luteolin inhibits H₂O₂-induced cell apoptosis by decreasing ROS levels and activating the PI3K/Akt pathway.¹⁸ Puerarin may play a neuroprotective role by activating the PI3K/Akt signaling pathway.¹⁹ Fucoidan can inhibit the apoptosis of PC12 cells induced by H₂O₂ by increasing the ratio of Bcl-2/Bax, reducing the expression of Caspase 3, and enhancing Akt phosphorylation.²⁰ A number of studies have shown that antioxidants participate in the PI3K/Akt pathway, which eventually protects PC12 cells from oxidative stress induced by H₂O₂. Therefore, in this study, to explore whether the PI3K/Akt signaling pathway was involved in hyperoside exerting its antioxidant effect on PC12 cells, we evaluated the expression levels of the flavonoid on the mRNA and key proteins involved in the PI3K/Akt signaling pathway. Therefore, in this study, in order to explore whether the PI3K/Akt signaling pathway is involved in the antioxidant effect of homoserin on PC12 cells, we evaluated the expression levels of flavonoids on mRNA and key proteins in the PI3K/Akt signaling pathway. Our results show that the expression of PI3K/Akt mRNA and P-PI3K/P-Akt protein after hyperoside treatment was significantly normalized. It indicates that the mechanism of homoserin reducing oxidative stress damage and inhibiting cell apoptosis may be through the PI3K/Akt signaling pathway.

As a transcription factor regulating the redox response, after activation, NF-κB can regulate gene expression in the early response of cell death induced by oxidative stress.²¹ NF-κB, as a signal element, controls the expression of the downstream inflammatory factor, nitric oxide synthase (iNOS).²² iNOS regulates cell apoptosis by synthesizing NO to influence the activation of NF-κB.²³Bax, as one of the apoptotic factors, can accelerate apoptosis. Caspase 3 protein, as a vital apoptotic executive protein in the middle and lower reaches of the Caspase cascade,²⁴ exists and plays an important role in apoptosis. NF-κB is a downstream factor of the Akt signaling pathway, and activated Akt can promote the expression of NF-κB protein. At the same time, NF-κB, as a key intermediate product of apoptosis induced by oxidative stress, also mediates Akt phosphorylation and gene expression. Therefore, activation of the PI3K/Akt and NF-κB pathways plays a functional role in bringing about anti-apoptotic effects. H₂O₂-induced oxidative stress injury increases NF-ĸB and Caspase 3 protein expression, which also results in an increase in apoptosis.^25,26 Studies report that H₂O₂ toxicity can increase the levels of NF-κB, Caspase 3, and the pro-apoptotic protein, Bax, while 6-(2-hydroxyethyl)-adenosine (HEA) treatment reduces NF-κB. The levels of Caspase 3 and Bax indicate that HEA can regulate the above pathways to reduce H₂O₂-induced cell apoptosis.²⁷ Numerous studies show that we can protect oxidative stress damage induced by H₂O₂ by reducing the activity of ROS and Caspase 3 in cells and by down-regulating the NF-κB pathway, thereby inhibiting cell apoptosis and inflammation.^28,29 Therefore, to elucidate further the anti-oxidant mechanism of hyperoside, we evaluated its effect on mRNA and protein expression of the NF-κB/Bax/Caspase 3 pathways. Our results showed hyperoside could reduce the expression level of NF-κB/Bax/Caspase 3 gene by regulating the NF-κB/Bax/Caspase 3 signaling pathway to decrease oxidative stress injury and protein expression levels; the decrease of its protein expression level indicates that the mechanism of hyperoside to reduce stress injury may be related to the signal pathway related to the regulation of inflammation and apoptosis by NF-KB/Bax/Caspase.

In addition to participating in the mediation of apoptosis-related pathways, the PI3K/Akt pathway can also regulate autophagy to eliminate protein aggregation in neurodegenerative diseases and regulate the autophagy proteins related to neurodegeneration. When autophagy occurs, LC3B, an important autophagy marker of the LC3 family, is recruited to autophagy. On the phage membrane, the multifunctional selective autophagy adaptor protein, P62, aggregates on the mitochondrial membrane through voltage-dependent anion channel protein 1 and specifically interacts with the ubiquitin-binding protein. Next, it binds to the LC3B protein and is selectively degraded in the autophagosome.²⁵Studies show that andrographolide activates autophagy and Nrf-2 mediated P62 signaling pathway-related autophagy genes and proteins (Beclin-1 and LC3), while reducing the expression of P-tau and P21 proteins, thereby protecting amyloid-induced and autophagy-related death in PC12 cells.³⁰ Studies have confirmed that the increase of P62 is closely linked to the unusual progress of autophagy flux and selectivity in the coaction environment stimulated by oxidative stress. In order to explore the protective action of hyperoside in H₂O₂-induced damage through autophagy-related pathways, our study evaluated the expression levels of this flavonoid on the mRNA and protein expression levels in the P62/LC3B pathway. The results showed that inhibition of autophagy after oxidative damage caused an increase in the expression and accumulation of the protein, P62, and the expression of LC3B was inhibited. After the interposition of hyperoside, the expression of P62 protein was significantly reduced, the expression of LC3B protein was activated, and the expression was significantly increased. It further indicates that the protective effect of hyperoside against H₂O₂ damage is achieved through autophagy-related pathways.

In summary, our results showed that hyperoside significantly up-regulated the gene and protein expression levels of PI3K, Akt, and LC3B. At the same time, the gene and protein expression levels of NF-κB, Bax, Caspase 3, and P62 showed a significant down-regulation trend, indicating that hyperoside could treat oxidative stress injury through the PI3K/Akt, NF-κB/Bax/Caspase 3, and P62/LC3B pathways .The above data indicate that hyperoside provides protection against oxidative stress induced by H₂O₂ in nerve cells through a variety of mechanisms. The protective effect of hyperoside in H₂O₂-induced injury was mainly achieved by regulating the pathways related to inflammation, autophagy, and apoptosis. Oxidative stress and apoptosis have a mutual regulatory effect on the activation and development of autophagy.^31,32 Oxidative stress, inflammation, apoptosis, and autophagy are inter-related and jointly regulate cell survival and growth. Hyperoside may be a promising therapeutic drug for the treatment of neurological diseases.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Natural Science Foundation of China [Grant number 81960701]; the Natural Science Foundation of Jiangxi Province [Grant number 20171ACB20039]; the Jiangxi Province talent Project [grant numbers (2016) 332]; Nanchang innovative talent team [Grant number (2018) 274]; Nanchang Key Laboratory of active ingredients of traditional Chinese medicine and natural medicine [Grant number 2019-NCZDSY-011].

ORCID iD

Yan Feng

References

Khanam

Ali

Asif

, . Neurodegenerative diseases linked to misfolded proteins and their therapeutic approaches: a review. Eur J Med Chem. 2016;124:1121-1141.doi:10.1016/j.ejmech.2016.08.006

27597727

Hui

Cuihong

. Research progress on the pathogenesis of neurodegenerative diseases. J Toxicol. 2006;32(06):59-63.doi:CNKI:SUN:WSDL.0.2018-06-016

Zhang

ZJ.

Cheang

LCV.

Wang

MW.

Lee

SM-Y.

Jun

Zhang

VLC

. Quercetin exerts a neuroprotective effect through inhibition of the iNOS/NO system and pro-inflammation gene expression in PC12 cells and in zebrafish. Int J Mol Med. 2011;27(2):195-203.doi:10.3892/ijmm.2010.571

21132259

Al-Ayadhi

. Oxidative stress and neurodegenerative disease. Neurosciences. 2004;9(1):19-23.doi:10.5772/28857

23377298

Zawia

NH.

Lahiri

DK.

Cardozo-Pelaez

. Epigenetics, oxidative stress, and Alzheimer disease. Free Radic Biol Med. 2009;46(9):1241-1249.doi:10.1016/j.freeradbiomed.2009.02.006

19245828

Glass

CK.

Saijo

Winner

Marchetto

MC.

Gage

. Mechanisms underlying inflammation in neurodegeneration. Cell. 2010;140(6):918-934.doi:10.1016/j.cell.2010.02.016

20303880

Kaewmool

Kongtawelert

Phitak

Pothacharoen

Udomruk

. Protocatechuic acid inhibits inflammatory responses in LPS-activated BV2 microglia via regulating SIRT1/NF-κB pathway contributed to the suppression of microglial activation-induced PC12 cell apoptosis. J Neuroimmunol. 2020;341:577164. doi:10.1016/j.jneuroim.2020.577164

32007785

Vila

Jackson-Lewis

Vukosavic

et al. Bax ablation prevents dopaminergic neurodegeneration in the 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson’s disease. Proc Natl Acad Sci U S A. 2001;98(5):2837-2842.doi:10.1073/pnas.051633998

11226327

Ling-Qun

Ji-ping

Qi-fu

et al. Study on the anti-apopotosis induced by hypoxia/hypoglycemia and reoxygenation of Panax notoginseng saponins in cultured rat hippocampal neurons. China J Chin Mater Med. 2003;28(1):52-55.doi:10.3321/j.issn:1001-5302.2003.01.020

10.

Galeotti

. Hypericum perforatum (St John’s wort) beyond depression: a therapeutic perspective for pain conditions. J Ethnopharmacol. 2017;200:136-146.doi:10.1016/j.jep.2017.02.016

28216196

11.

Haiyan

Liwen

. Mitochondrial dysfunction and the pathogenesis of epilepsy. J Apop Nerv Dis. 2018;35(08):766-768.doi:CNKI:SUN:ZFSJ.0.2018-08-026

12.

X-C.

W-L.

Liu

H-C.

Jiang

Y-P

. Protective effect of hyperoside against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells. Artif Cells Nanomed Biotechnol. 2020;48(1):377-383.doi:10.1080/21691401.2019.1709851

31903787

13.

Fan

H-H.

Zhu

L-B.

et al. Hyperoside inhibits lipopolysaccharide-induced inflammatory responses in microglial cells via p38 and NFκB pathways. Int Immunopharmacol. 2017;50:14-21.doi:10.1016/j.intimp.2017.06.004

28622577

14.

Jang

JH.

Surh

. Protective effects of resveratrol on hydrogen peroxide-induced apoptosis in rat pheochromocytoma (PC12) cells. Mutat Res. 2001;496(1-2):181-190.doi:10.1016/S1383-5718(01)00233-9

11551494

15.

Liu

Tao

Zhang

Wei

. Protective effects of hyperoside (quercetin-3-o-galactoside) to PC12 cells against cytotoxicity induced by hydrogen peroxide and tert-butyl hydroperoxide. Biomed Pharmacother. 2005;59(9):481-490.doi:10.1016/j.biopha.2005.06.009

16271843

16.

Rickle

Bogdanovic

Volkman

Winblad

Ravid

Cowburn

. Akt activity in Alzheimer’s disease and other neurodegenerative disorders. Neuroreport. 2004;15(6):955-959.doi:10.1097/00001756-200404290-00005

15076714

17.

Qin

Yang

et al. Neuroprotective effects of deuterium-depleted water (DDW) against H₂O₂-induced oxidative stress in differentiated PC12₂cells through the PI3K/Akt signaling pathway. Neurochem Res. 2020;45(5):1034-1044.doi:10.1007/s11064-020-02978-4

32016793

18.

Lin

Tian

X-H.

Y-S.

Jiang

W-S.

Zhou

Y-J.

Cheng

W-J

. Luteolin-induced protection of H₂O₂-induced apoptosis in PC12 cells and the associated pathway. Mol Med Rep. 2015;12(5):7699-7704.doi:10.3892/mmr.2015.4400

26459830

19.

Zhang

Huang

W-D.

X-Y.

Yang

Y-M

. Puerarin protects differentiated PC12 cells from H₂O₂-induced apoptosis through the PI3K/Akt signalling pathway. Cell Biol Int. 2012;36(5):419-426.doi:10.1042/CBI20100900

22126839

20.

Gao

Dong

Yin

Shen

Tian

. Neuroprotective effect of fucoidan on H₂O₂-induced apoptosis in PC1₂ cells via activation of PI3K/Akt pathway. Cell Mol Neurobiol. 2012;32(4):523-529.doi:10.1007/s10571-011-9792-0

22222440

21.

Lee

SY.

Lee

JW.

Lee

et al. Inhibitory effect of green tea extract on beta-amyloid-induced PC12 cell death by inhibition of the activation of NF-kappaB and ERK/p38 MAP kinase pathway through antioxidant mechanisms. Brain Res Mol Brain Res. 2005;140(1-2):45-54.doi:10.1016/j.molbrainres.2005.07.009

16153742

22.

Yanping

Wang Yang

et al. Oxidative stress and autophagy. J Anim N. 2016;28(9):2673-2680.doi:10.3969/j.issn.1006-267x.2016.09.002

23.

Xiaohong

Yueyang

Ziwei

et al. Research progress of traditional Chinese medicine in regulating cerebral ischemia MAPK signaling pathway. CHN Med Her. 2019;16(04):47-50.doi:CNKI:SUN:YYCY.0.2019-04-012

24.

Ooi

KL.

Loh

SI.

Sattar

MA.

Muhammad

TST.

Sulaiman

et al. Cytotoxic, caspase-3 induction and in vivo hepatoprotective effects of phyllanthin, a major constituent of Phyllanthus niruri . J Funct Foods. 2015;14:236-243.doi:10.1016/j.jff.2015.01.032

25.

Jingdong

. Oxidative stress and related diseases and their mechanism of action. J Shenyang Med Colg. 2018;20(03):272-276.doi:10.16753/j.cnki.1008-2344.2018.03.022

26.

Wang

et al. Ginsenoside Rd attenuates myocardial ischemia/reperfusion injury via Akt/GSK-3β signaling and inhibition of the mitochondria-dependent apoptotic pathway. PLoS One. 2013;8(8):e70956. doi:10.1371/journal.pone.0070956

23976968

27.

Zhang

Olatunji

OJ.

Tang

Wei

Ouyang

. N6-(2-hydroxyethyl)-adenosine from Cordyceps cicadae attenuates hydrogen peroxide induced oxidative toxicity in PC12 cells. Metab Brain Dis. 2019;34(5):1325-1334.doi:10.1007/s11011-019-00440-1

31197679

28.

Wang

et al. Neuroprotective effects of macranthoin G from Eucommia ulmoides against hydrogen peroxide-induced apoptosis in PC12 cells via inhibiting NF-κB activation. Chem Biol Interact. 2014;224:108-116.doi:10.1016/j.cbi.2014.10.011

25451577

29.

Liu

Han

et al. Potent effects of flavonoid-rich extract from Rosa laevigata Michx fruit against hydrogen peroxide-induced damage in PC12 cells via attenuation of oxidative stress, inflammation and apoptosis. Molecules. 2014;19(8):11816-11832.doi:10.3390/molecules190811816

25105919

30.

Zhang

. Andrographolide protects PC12 cells against β-amyloid-induced autophagy-associated cell death through activation of the Nrf2-mediated p62 signaling pathway. Int J Mol Sci. 2018;19(9):2844. doi:10.3390/ijms19092844

31.

Ahlawat

Sharma

. A new promising simultaneous approach for attenuating type II diabetes mellitus induced neuropathic pain in rats: iNOS inhibition and neuroregeneration. Eur J Pharmacol. 2018;818:419-428.doi:10.1016/j.ejphar.2017.11.010

29154836

32.

Yang

Xiaochen

Junping

. New progress in research on the role of oxidative stress and autophagy in atherosclerosis. Chin J Arteriosclerosis. 2017;25(04):404-410.doi:10.3969/j.issn.1007-3949.2017.04.016

Protective Effects and Mechanism of Hyperoside in PC12 Cells Against Oxidative Stress Injury Induced by Hydrogen Peroxide

Abstract

Keywords

Materials and Methods

Reagents and Drugs

Cell Culture and Modeling

Cell Viability Assays

Reverse Transcription PCR (RT-PCR)

Western Blot (WB) Analysis

Statistical Analysis

Results

Protective Effect of Hyperoside on PC12 Cell Viability After H2O2 Treatment

Increased PI3K and Akt mRNA and Its Phosphorylated Protein Expression in Hyperoside-Treated Cells

Changes in NF-κB/Bax/Caspase 3 mRNA and phosphorylated protein expression in hyperoside-treated cells

Changes in P62/LC3B mRNA and Phosphorylated Protein Expression in Hyperoside-Treated Cells

Discussion

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iD

References

Protective Effect of Hyperoside on PC12 Cell Viability After H₂O₂ Treatment