How Do Phenylethanoid Glycosides, The Active Ingredient Of Cistanche, Play A Neuroprotective Role?

Xue Gong†a , Yan Xu†b , Kai Renc , Xiaorong Baid , Chunhong Zhanga

ABSTRACT In this study, we isolated eight phenylethanoid glycosides from Paraboea martini for the first time and evaluated the mechanism underlying their neuroprotective effects against H2O2- induced injury in PC12 cells. The MTS method was utilized to screen the phenylethanoid glycosides for protective ability. Next, qRT-PCR and western blotting analysis were used to detect the transcription levels of HO-1 and GCLC, which are regulated by Nrf2. The inhibitor ZnPP was used to analyze the involvement of Nrf2 in HO-1 expression. Analyses showed that caleolarioside B, paraboside B, and paraboside II also upregulated the expression of HO-1, but showed no obvious effect on GCLC. Pretreatment with ZnPP significantly reduced the neuroprotective effects. Thus, phenylethanoid glycosides isolated from P. martini protected PC12 cells from H2O2-induced damage by upregulating HO-1. The results provided evidence that P. martini might be a potential therapeutic agent for the treatment of neurodegenerative diseases.

ARTICLE HISTORY Received 8 April 2019 Accepted 30 July 2019

KEYWORDS Paraboea martini; phenylethanoid glycosides; neuroprotective effects; zinc protoporphyrin; HO-1

Contact: joanna.jia@wecistanche.com / WhatsApp: 008618081934791

The mechanism of the neuroprotective effect of Cistanche deserticola, click here to learn more

Oxidative stress is known to play an essential role in the pathogenesis of several diseases. It is linked to the etiology of Alzheimer’s disease (AD), Parkinson’s disease (PD), epilepsy, and other diseases [1–3]. At present, continuing progress has been made in the study of the pathogenesis of AD, PD, and other diseases. However, because of the limited knowledge of the molecular mechanisms underlying AD, PD, and other diseases, effective preventive or curative strategies for these diseases have not been realized [4]. H2O2 is an important component of reactive oxygen species (ROS). Accordingly, H2O2-induced PC12 cell injury is the most commonly used model of oxidative stress and is widely used in experimental studies of neuroprotective effects [5]. Heme oxygenase 1 (HO-1) and glutamate-cysteine ligase-catalytic subunit (GCLC) are important cellular antioxidant enzymes, and both genes are regulated downstream of nuclear factor erythroid 2 (Nrf2), which is emerging as a promising therapeutic target for neuroprotection [6,7]. Phenylethanoid glycosides, derived from the C-6-C-3 unit, contain many phenolic hydroxyl groups and have significant antioxidant and antiaging activities. Over the past few years, studies have indicated that many medicinal plants contain phenylethanoid glycosides that show significant antioxidant activity. For example, phenylethanoid glycosides derived from Herba cistanche are known to exert protective effects on cognitive deficits in AD. That study investigated the associated protective mechanism using an AD senescence-accelerated prone mouse 8 (SAMP8) model. The results indicated that the ability of phenylethanoid glycosides to ameliorate cognitive deficits in SAMP8 mice might be related to the promotion of synaptic plasticity involving antioxidant processes [8]. In this study, two phenylethanoid glycosides were separated and purified from Tectona grandis (teak) wood knots; these compounds displayed notable antioxidant effects that were determined using an amperometric method [9]. Further, caffeoyl phenylethanoid glycoside compounds from Lindernia ruellioides exert dose-dependent antioxidant and superoxide anion-free radical-scavenging effects in vitro [10]. The mechanisms of action of phenylethanoid glycosides are related to the regulation of the Nrf2/ARE signaling pathway, which influences the synthesis of SOD, CAT, GSH-PX, and other antioxidant enzymes [11]. The family Gesneriaceae, which contains 150 genera and approximately 3700 species, is primarily distributed in the tropical and temperate regions of eastern and southern Asia, Oceania, South America, Africa, Southern Europe, and Mexico [12]. There are 54 genera and approximately 463 species belonging to Gesneriaceae in China. It was reported that approximately 100 species have medical efficacy and most of these are mainly distributed in Guangxi and Guizhou provinces [13]. In recent years, research on Gesneriaceae plants has gradually increased in different areas of the world, and some new constituents with physiological activities have been found [14]. Paraboea martinii (Levl.) Burtt belongs to Parabola (Gesneriaceae) and is an herbaceous plant. Based on “The Chinese Traditional Medicine Resource Records”, it was recorded that the whole plant is used as medicine. Moreover, it can exert many pharmacological activities on conditions such as hematemesis, edema, dysentery, traumatic injury, and fracture [15]. Bai proved that phenylethanoid glycosides are one of the main chemical constituents of Gesneriaceae plants [16]. Some researchers have also found that phenylethanoid glycosides have significant antioxidant activities [17]. Li demonstrated that phenylethanoid glycosides of Cistanche deserticola have significant antioxidant activity [18]. In addition, Ju systematically studied the neuroprotective activities of phenylethanoid glycosides using PC12 cell models. The results indicated that phenylethanoid glycosides significantly attenuate the damage induced by Aβ1-42 [19]. In our studies, we isolated eight phenylpropanoid glycosides (paraboside A, paraboside B, paraboside I, paraboside II, paraboside III, nuomioside A, caleolarioside B, isonuomioside A) from P. martini [20]. All compounds were isolated from the genus Parabola for the first time, and the compounds paraboside A, paraboside B, p paraboside I, paraboside II, and paraboside III were new compounds [20]. In a continuing search for molecules with antioxidant properties, phenylpropanoid glycosides were examined. However, whether these phenylpropanoid glycosides from P. martini could protect against H2O2-induced PC12 cell injury was previously unclear. In this study, we examined the protective effects of phenylpropanoid glycosides on H2O2-induced damage to PC12 cells. We also examined the effects and preliminary mechanism underlying the activity of phenylpropanoid glycosides with respect to the activation of HO-1 and GCLC in vitro. Materials and methods Materials Paraboea martini (Level.) Burtt was collected from Daxin County, Guangxi Autonomous Region in August 2014 and verified by Dr. Minhui Li (Baotou Medical College). Voucher specimens were deposited at the Laboratory of Pharmacognosy and Phytochemistry. Eight phenylpropanoid glycosides (paraboside A, paraboside B, paraboside I, paraboside II, paraboside III, nuomioside A, caleolarioside B, isonuomioside A) were extracted and separated from P. martinii (Levl.) Burtt by prof. Xiaoqin Wang [20]. The purity (> 98%) of eight phenylpropanoid glycosides was measured by the HPLC peak area normalization method. As an example, the chromatography charts of paraboside B and paraboside II are shown in Figure 1, and their relative content was found to be 98.23% and 99.20%, respectively. The poorly-differentiated rat adrenal pheochromocytoma cell line (PC12) was purchased from the Cell Bank at the China Academy of Science (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM) and horse serum were purchased from Gibco (Gaithersburg, MD, USA). Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Hyclone, Waltham, MA, USA). H2O2 was obtained from Tianjin Fu Yu Reagent Co., Ltd. (Tianjin, China). Trizol was purchased from Invitrogen (Carlsbad, CA, USA). PrimeScriptTM RT reagent Kit (Perfect Real Time) was obtained from TaKaRa Biotechnology Co. (Dalian, China). Primers for HO-1, GCLC, and GAPDH were synthesized by Sangon Biotech (Shanghai, China). The CellTiter 96® Aqueous One Solution Cell Proliferation Assay kit (MTS) was obtained from Promega Corp. (Madison, USA). Inhibitors of HO-1 zinc protoporphyrin (ZnPP) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). A Thermo311 CO2 Incubator (Thermo, USA), StepOnePlusTM Real-Time PCR Instrument Thermal Cycling Block (Thermo), Thermo Scientific Multiskan FC (Thermo), and xCELLigence RTCA DPlus Analyzer and E-Plate 16 (ACEA Biosciences, USA) were also used.

Cell culture and treatment

PC12 cells were grown in DMEM containing 5% FBS and 5% horse serum at 37°C in a humidified 5% CO2 atmosphere. PC12 cells were divided into three groups as follows: a control group, a model group, and a treatment group. Eight phenylpropanoid glycosides were dissolved in DMSO (< 0.01%) and then diluted with a complete culture medium. To study the effects of the eight phenylpropanoid glycosides on H2 O2-induced cell injury, PC12 cells were cultured with the eight compounds at concentrations of 3.125, 6.25, 12.5, 25, and 50 μM for 12 h.

Determination of H2O2

model, We determined the appropriate concentration of H2 O2 using real-time cellular analysis (RTCA). PC12 cells were seeded in a CIM-plate-16 microplate at a volume of 100 µL and then incubated for 12 h at 37°C in an atmosphere of 5% CO2. The PC12 cells were divided into control and model groups. Next, 10 μL H2O2 (50, 100, 200, and 400 μM) was added to the model groups, and the control groups were treated with 10 μL complete DMEM, set up in three complex wells. The E-Plate 16 was placed in the xCELLigence RTCA, monitored every 15 min, and the results were recorded for 32 h. The effect of H2O2 on PC12 cells was analyzed using the xCELLigence RTCA data analysis software. The cell index (CI) value was used to determine the optimal H2O2 concentration and duration of action.

Compound extraction and isolation

Fractions were separated based on their polarity using previously described methods. Air-dried, powdered P. martinii (4 kg) was successively extracted three times with a 75% aqueous ethanol solution [20]. The obtained extracts were then concentrated using a rotary evaporator to remove ethanol, and crude extracts were obtained. The samples were dissolved in deionized water and then extracted successively with petroleum ether, ethyl acetate, n-butanol, and water. The n-butanol extract (400 g) was fractionated using an AB-8 macroporous resin column and an ethanol-H2O2 solution (0, 10, 30, 50, 70, and 95%). Six fractions (fractions 1–6) were finally obtained. Fractions 3 and 4 were subjected to separation using an MCI column with a step-gradient of MeOH-H2O2 (10–100%). Next, the products were isolated using Sephadex LH-20 with 50% MeOH as the eluent. Compounds 1 (41.0 mg) and 6 (15.6 mg) were isolated from fraction 3. Compounds 2 (62.0 mg), 3 (34.7 mg), 7 (11.0 mg), and 8 (26.0 mg) were obtained from fraction 4 using Sephadex LH-20 chromatography with 50% MeOH as the eluent. Final isolations were performed by semi-prep, reversed-phase HPLC (Merck LiChrosorb, RP-18, RP-18, 250 × 10 mm) with 50% MeOH and UV detection at 280 nm to yield compounds 4 (39.0 mg) and 5 (9.0 mg) [20].

Cell viability assay

PC12 cells were seeded in 96-well plates at a density of 2 × 104 cells/well at a final volume of 100 µL. PC12 cells were incubated at 37°C with 5% CO2 for 12 h. Next, the cells were treated with various concentrations of crude extracts (0.025, 0.05, 0.1, 0.2, and 0.4 mg/mL) and compounds 1, 2, 3, 4, 5, 6, 7, and 8 at five concentrations (3.125, 6.25, 12.5, 25, and 50 μM). After pretreatment for 24 h, cell viability was determined by MTS assays. PC12 cells were preincubated with or without the HO-1 inhibitor without ZnPP (15 μM) for 30 min, then incubated with or without caleolarioside B, paraboside B, and paraboside II (3.125 μM) for 12 h, and finally incubated with 400 μM H2O2 for another 6 h. After treatment, the number of surviving cells was determined by an MTS assay [6].

Protective effects of crude extracts and monomeric compounds on H2O2-treated PC12 cells

The cells were cultured in a 96-well plate at a final volume of 100 µL. Serial dilutions of crude extracts (0.025, 0.05, 0.1, 0.2, and 0.4 mg/mL) were added to the treated group. After pretreatment for 12 h, 400 μM of H2O2 solution (optimal concentration) was added to the treated group and the H2O2 group of each well for 6 h. MTS solution was then added to each well, and the plates were incubated for 3 h at 37° C. The plates were oscillated at low speed for 5 min at room temperature until all crystals were fully dissolved. Cell protection was determined based on MTS assay results according to the manufacturer’s 2204 X. GONG ET AL. Downloaded from https://academic.oup.com/bbb/article/83/12/2202/6044145 by guest on 27 August 2021instructions. The optical density was determined at an absorbance wavelength of 490 nm. We further tracked the activity of compounds 1, 2, 3, 4, 5, 6, 7, and 8 isolated from fractions 3 and 4. Serial dilutions of compounds 1, 2, 3, 4, 5, 6, 7, and 8 (3.125, 6.25, 12.5, 25, and 50 μM) were added to the treated group to determine their respective protective effects on H2O2-induced PC12 cells.

Quantitative real-time PCR (qRT-PCR)

PC12 cells were harvested at 12 h after treatment with 3.125 μM caleolarioside B, paraboside B, and paraboside II. Total RNA was extracted using TRIzol-reagent, and aliquots of total RNA were reverse-transcribed using a Primescript RT Master Kit according to the manufacturer’s instructions. The following primers were employed for the experiments in accordance with a previous report [21]. HO-1: Forward 5′ -GC CTGCTAGCCTGGTTCAAG-3′, Reverse 5′ -AGC GGTGTCTGGGATGAACTA-3′; GCLC: Forward 5′ - GTCCTCAGGTGACATTCCAAGC-3′, Reverse 5′ -TG TTCTTCAGGGGCTCCAGTC-3′; GAPDH: Forward 5′-AAGCTGGTCATCAACGGGAAAC-3′, Reverse 5′- GAAGACGCCAGTAGACTCCACG-3′. Aliquots of the obtained cDNAs were then amplified by PCR. The reaction conditions were as follows: initial denaturation at 95°C (30 s), followed by 40 cycles of at 95°C (5 s), 60°C (34 s), and 72°C (35 s). All primers were tested and the FL fluorescent signal was detected; then, the △Ct value was calculated and the relative values were compared to those of the control group using the following equation: △Ct = Ct (sample) − Ct (endogenous control), △△Ct = △Ct (sample) −△Ct (untreated), and fold change = 2−△△Ct. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.

Protein extraction

PC12 cells were seeded on 100-mm dishes at 1 × 107 cells/dish. After attachment, the cells were respectively treated with caleolarioside B, paraboside B, and paraboside II (3.125 µM) for 12 h. The glycosides were then removed, and the cells were incubated with H2O2 for another 6 h. After incubation, the cells were washed twice with cold PBS and scraped from the dishes with 1 mL of PBS. Cell homogenates were centrifuged at 1500 rpm for 10 min. After treatment, cellular proteins were extracted using a Beyotime RIPA cell lysis buffer with 1 mM PMSF according to the manufacturer’s instructions. In addition, cytoplasmic and nuclear proteins were isolated as described in the Beyotime nuclear and cytoplasmic extraction kit protocol. The protein concentrations of the samples were detected using a Beyotime BCA protein assay kit, and all samples were stored at −80°C for western blotting analysis [6].

Western blot analysis

SDS-PAGE electrophoresis was performed after extraction and subsequent denaturation of total protein from different groups. After electrophoresis, the protein was transferred to PVDF membranes, which were then blocked with 5% skim milk powder for 4 h. After washing, primary antibody (rabbit polyclonal antibody GAPDH (1:10,000 dilution), rabbit polyclonal antibody HO-1 (1:1000 dilution), or rabbit polyclonal antibody GCLC (1:1000 dilution) was added to the membranes, which were incubated overnight at 4°C. The next day, the PVDF membranes were washed three times in TTBS. Next, secondary antibody (conjugated affinity pure goat anti-rabbit IgG (1:1000 dilution) labeled with horseradish peroxidase was added to the membranes, which were then incubated at room temperature for 2 h. The PVDF membranes were washed and subjected to ECL chemiluminescence detection reagent for signaling exposure on X-ray film, which was then fixed and scanned [22].

Statistical analysis

The data were analyzed using SPSS 19.0, and the results are presented as the means with standard deviation (SD) of three independent experiments. Values of p < 0.05 were considered to indicate statistically significant differences.

Results Establishment of H2O2 model

As shown in Figure 2, H2O2 at 50, 100, and 200 μM did not cause the appropriate level of cell death. However, 400 μM H2O2 acted on PC12 cells within 2–6 h, and the oxidative damage to cells tended to be stable. Therefore, we considered that induction with H2O2 at a concentration of 400 μM for 6 h was appropriate for mimicking oxidative stress-induced injury to PC12 cells.

Protective effects of crude extracts on PC12 cells treated with H2O2

Compared to that in the H2O2 model group (Figure 3), n-butanol extract at concentrations of 0.05, 0.1, 0.2, and 0.4 mg/mL significantly improved oxidative damage to PC12 cells induced by H2O2 (p < 0.05) in a concentration-dependent manner. These data suggested that the n-butanol fraction contained more potent protective activity. However, petroleum ether and ethyl acetate extracts showed protective activity only at high concentrations (petroleum ether: 0.1 and 0.2 mg/ mL, p < 0.05; ethyl acetate: 0.2 and 0.4 mg/mL, p < 0.05). Moreover, different concentrations of aqueous phase extracts showed no protective effects (p > 0.05). Furthermore, we have added data from a validation study using SH-SY5Y human neuroblastoma cell lines (SH-SY5Y) in the Supplemental File. Treatment with 250 μM H2O2 resulted in decreased cell viability; however, caleolarioside B, paraboside B, and paraboside II (3.125, 6.25, 12.5, 25, and 50 μM) treatment significantly attenuated SH-SY5Y cell death. The experiment results proved that caleolarioside B, paraboside B, and paraboside II protected SHSY5Y cells from H2O2-induced cell injury.

Structures of compounds isolated from the n-butanol fraction

To confifirm that the n-butanol extract contained active components, we further fractionated eight compounds from the extraction. The structures of compounds 1, 2, 3, 4, 5, 6, 7, and 8 were identifified by difffferent spectral data (1 H-NMR, 13C-NMR, and ESI-MS) based on comparisons to published data for paraboside A, paraboside B, paraboside I, paraboside II, paraboside III, nuomioside A, caleolarioside B, and isonuomioside A, respectively [20]. For example, the data for paraboside B were as follows: brown gum; ½α 20 D −55.7 (c 0.05, CH3OH) [20]; UVMeOH max nm (logε): 220 (3.61), 291 (3.54), 330 (3.66) [20]; IRKBr max cm−1 : 3392, 2943, 2885, 1699, 1683, 1606, 1521, 1361, 1282, 1163, 1114, 1039, 995, 817 cm−1 [20]; for 1 H and 13C NMR spectroscopic detailed data shown in reference 20; HR-ESI-MS (negative) m/z 741.2231 [M-H]− (calcd. for C33H41O19, 741.2242) [20]. The data for paraboside II were as follows: white amorphous powders; ½α 20 D −80.5 (c 0.05, CH3OH) [20]; UVMeOH max nm (logε): 229 (3.74), 322 (3.73) [20]; IRKBr max cm−1 : 3419, 1699, 1625, 1596, 1515, 1456, 1419, 1263, 1159, 1139, 1068, 1022, 808 cm−1 [20]; for 1 H and 13C NMR spectroscopic detailed data shown in reference 20; ESI-MS (negative) m/z 637

Protective effects of isolated compounds against H2 O2-induced toxicity in PC12 cells

MTS assays were used to investigate the effect of isolated compounds on the viability of PC12 cells exposed to H2O2-induced toxicity. As shown in Figure 5, compared to that in the control group, PC12 cells pretreated with five different concentrations of caleolarioside B, paraboside B, paraboside II, or paraboside A (3.125, 6.25, 12.5, 25, and 50 μM) for 24 h showed no significant differences in cell viability (p > 0.05). Thus, pretreatment of PC12 cells with caleolarioside B, paraboside B, and paraboside II resulted in no cytotoxicity. As shown in Figure 6, 400 µM H2O2 significantly decreased cell viability compared to that in the control group. Moreover, the cell viability of H2O2- treated PC12 cells markedly increased following pretreatment of PC12 cells with different concentrations of caleolarioside B, paraboside B, and paraboside II; furthermore, cell viability was the highest following treatment with 50 µM caleolarioside B, 50 µM paraboside B, and 12.5 µM paraboside II. The other five compounds showed no significant protective effects on H2O2-treated PC12 cells. To illustrate this finding, we discuss one of these as an example, whereas the others are not described in the manuscript.

Transcription levels of HO-1 and GCLC

We next tested the transcriptional levels of HO-1 and GCLC in PC12 cells treated with caleolarioside B, paraboside B, and paraboside II. Total mRNA was collected from treated and untreated PC12 cells for 12 h, and then subjected to expression analysis by qRT-PCR. As shown in Figure 7(a,c,e), the transcriptional levels of HO-1 significantly increased after

PC12 cells were pretreated with the indicated concentrations of caleolarioside B, paraboside B, and paraboside II for 12 h (p < 0.05). As shown in Figure 7(b), the transcriptional levels of GCLC also significantly increased in PC12 cells pretreated with the indicated concentrations of caleolarioside B for 12 h. However, levels of GCLC in PC12 cells pre-treated with paraboside B for 12 h decreased significantly compared to those in the model group (p < 0.05; Figure 7(d)). Furthermore, with paraboside II treatment, the levels of GCLC in PC12 cells were not significantly different compared to those in the model group (p > 0.05; Figure 7(f)).

Expression of HO-1 and GCLC

HO-1 and GCLC are important cellular antioxidant enzymes, and both are Nrf2-regulated downstream genes. The protein expression of HO-1 and GCLC was also observed after treatment [6]. Figure 8 shows the protein levels of HO-1 and GCLC in PC12 cells after treatment with 400 μM H2O2; the results indicated that the FL fluorescent intensities of HO-1 in the caleolarioside B and paraboside B groups were greater than those in the H2O2-treated groups (Figure 8(a,c)). However, the expression of GCLC was not significantly changed by the compounds, except for caleolarioside B (Figure 8(b)). Next, chemical inhibitors of HO-1 were used to further evaluate the roles of antioxidant enzymes in regulating the protective effects exerted by caleolarioside B, paraboside B, or paraboside II against H2O2-induced cytotoxicity. Caleolarioside B, paraboside B, and paraboside II (3.125 µM) prevented H2O2-induced cytotoxicity, but such protective effects were reversed by the HO-1 inhibitor ZnPP (p < 0.01) at 15 µM (Figure 8(d)).

Discussion

Population aging is a severe global problem. Many diseases are related to senility, such as AD and PD, which are serious threats to public health. AD and PD have emerged as the third most lethal diseases globally, after cardiovascular disease and cancer. Therefore, neurodegeneration has become an urgent research topic worldwide. However, the pathogenesis of AD and PD, neurodegenerative diseases of the central nervous system, has remained unclear to date. Furthermore, there are only a few drugs that can effectively treat AD or PD because of their complex pathogenic mechanisms [23]. Previous studies showed that ROS, such as superoxide anion (O2−) and H2O2, are the main factors contributing to neurodegenerative diseases. Pathological changes during PD include neuronal degeneration and death of the substantia nigra and striatal dopamine [24,25]. Accordingly, it is important to develop effective antioxidant drugs to treat PD, and such drug discovery has become an important research direction [26]. Currently, the Nrf2/ARE signaling pathway appears to be the most important endogenous antioxidant stress pathway [27]. AREs are located in the 5′- flflanking regions of genes, such as HO-1 and GCLC [28,29]. HO-1 and GCLC are Nrf2-related genes that are regulated downstream of this protein [6]. AREs can subsequently affect the levels of antioxidants and activate the downstream expression of HO-1 and other protective genes, which could enhance cell protection activities [30,31]. Furthermore, findings suggested that the protective effects of resveratrol against Aβ1-42- induced toxicity in PC12 cells occur through the upregulation of HO-1 expression via activation of the Nrf2 intracellular signaling pathway [32].

PC12 cells are an accepted model of neural cells and are extensively used in neuroprotective effect studies [33]. H2O2 can easily pass through the cell membrane and combine with iron to form highly-active free radicals, which induce oxidative stress and cell injury [34]. H2O2 is an important ROS, and free radicals are easily generated and relatively stable, thus, they are usually used to establish an oxidative stress model using PC12 cells [5]. In this study, pretreatment of PC12 cells with nontoxic concentrations of the plant extract protected cells from H2O2-induced cytotoxicity by decreasing the generation of ROS. Phenylethanoid glycosides contain many phenolic hydroxyl groups that have antioxidant and antiaging activities. In this study, we found that n-butanol extraction produced better protection than other polarity fractions (Figure 3). Thus, we further screened the protective activities of eight compounds isolated from the n-butanol fraction. The results showed for the first time that Figure 8. Analysis of HO-1 and GCLC protein levels on PC12 cells. (a) Western blotting analysis of the expression of HO-1 and GCLC and GAPDH was used as a loading control. (b) Western blot analysis GCLC protein expression. (c) Western blot analysis HO-1 protein expression. (d) HO-1 inhibitor ZnPP reduced the neuroprotective effect. Data are presented as means ± SD, n = 3. ***p < 0.001 versus control group; #p < 0.05 versus H2O2 group (without ZnPP treated), ##p < 0.01 versus H2O2 group (without ZnPP treated), ###p < 0.001 versus H2O2 group (without ZnPP treated); &&p < 0.01, versus H2O2 group (with ZnPP treated), &&&p < 0.001, versus H2O2 group (with ZnPP treated). (Control group; Model group: H2O2; ZnPP negative group: caleolarioside B + H2O2, paraboside B + H2O2, paraboside II + H2O2; ZnPP positive group: ZnPP + caleolarioside B + H2O2, ZnPP + paraboside B + H2O2, ZnPP + paraboside II + H2O2). 2210 X. GONG ET AL. Downloaded by a guest on 27 August 2021caleolarioside B, paraboside B, and paraboside II have significant protective effects against H2O2- induced oxidative damage in PC12 cells. The results also indicated that H2O2 treatment induced significant PC12 cell injury and decreased cell viability, whereas caleolarioside B, paraboside B, and paraboside II treatment significantly mitigated these effects (Figure 6). A previous study reported that the Nrf2/ARE pathway is one of the most important endogenous antioxidant pathways, and can upregulate the expression of downstream markers, such as HO-1, to protect cells [7]. The results of qRT-PCR and western blotting analyses showed that caleolarioside B, paraboside B, and paraboside II significantly upregulated the levels of HO-1. However, the transcriptional levels of GCLC significantly increased in PC12 cells pretreated with caleolarioside B but decreased in those pretreated with paraboside B compared to those in the model group. Further, arabinoside II did not alter the expression of this marker, compared to that in the model group (Figures 7 and 8(a–c)). In addition, the protective effects of caleolarioside B, paraboside B, and paraboside II against H2O2-induced PC12 cell injury were reversed by the HO-1 inhibitor ZnPP (Figure 8(d)). In conclusion, the phenylethanoid glycosides caleolarioside B, arabinoside B, and paraboside II effectively protected PC12 cells from H2O2-induced oxidative damage. Accordingly, these compounds, isolated from P. martini, might represent potential drug candidates for the treatment of neurodegenerative diseases.

Author contributions L. M., and Z. C. conceived and designed the experiments. G. X., R. K., and B.X. performed the experiments. G. X. and X. Y. wrote the paper. G. X., X. Y., R.K., B. X., Z. C., and L. M. discussed the results, commented on the manuscript. All authors read and approved the final manuscript.

Disclosure statement No potential conflict of interest was reported by the authors. Funding This work was supported by Research Funds of Baotou Medical College [grant number: BYJJ-YF 201727], and the 2018 Chinese medicine public health service subsidy special “the fourth survey on Chinese materia medica resource” [grant number: Finance Society [2018] 43].

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