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The potential antioxidant ability of hydroxytyrosol on Caenorhabditis elegans against oxidative damage via the insulin signaling pathway
⁎Corresponding author. dcb@sicau.edu.cn (Chun bang Ding)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Abstract
Hydroxytyrosol (HY) deriving from olive leaves is a phenolic component which has been proven to possess a strong antioxidant ability. However, the underling mechanism is still unclear. To evaluate the antioxidant ability of HY comprehensively, assays in vitro and in vivo (Caenorhabditis elegans (C. elegans) was used as a model organism) were conducted. The results showed HY could scavenge 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) radicals with a strong total reducing power. Pretreated with HY for 48 h, the cell viability of Chinese hamster ovary (CHO) cells was enhanced under oxidative stress by reducing the level of reactive oxygen species (ROS) and malondialdehyde (MDA). A suitable concentration of HY showed no side effects on the development, fertility, and movement of C. elegans. With the treatment of HY, the survival was enhanced by 15.79% under thermal stress. The ROS and MDA contents were also reduced, which might be associated with the increasing abilities of antioxidant enzymes such as superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) and the heat shock protein HSP-16.2. Nuclear localization of DAF-16 was greatly increased after treated with HY. More outcomes demonstrated HY exhibited an excellent antioxidant capacity via the insulin signaling pathway by upregulating daf-16 and sod-3 and downregulating the genes of age-1 and daf-2.
Keywords
Hydroxytyrosol
Caenorhabditis elegans
CHO cells
Antioxidant ability
1 Introduction
Olive leaves are used to treat influenza, diarrhoea, malaria, dengue fever and to prevent urinary and surgical infections with remarkable effect because of its abundant chemical composition in the Mediterranean (Bock et al., 2013; Şahin and Bilgin, 2018). The main active components include secoiridoids, flavonoids, phenolic acids and simple phenols. The significant biological activity and potential pharmacological effects of olive leaves is mainly due to the abundant phenolic substances, especially hydroxytyrosol (HY) (3,4-dihydroxyphenylethanol). The molecular formula of HY was C8H10O3 with a relative molecular weight of 154.16. The beneficial effects of HY comprise multi-target activities including the antimicrobial, antineoplastic, hypoglycemic effects as well as prevention of cardiovascular and cerebrovascular diseases. Ghalandari and colleagues carried out the antibiogram test of HY and its two lipophilic derivatives hydroxytyrosol acetate and hydroxytyrosol oleate by using well assay and Microplate broth dilution for Staphylococcus aureus and Staphylococcus epidermidis and HY showed the strongest antibacterial effect with MIC of 3.13 and 6.25 mg/mL against the above two strains, respectively (Ghalandari et al., 2018). And HY also showed an inhibitory effect on Propionibacterium acnes with MIC and MBC of 6.25 and 12.5 mg/mL, respectively (Owrang et al., 2017). HY could also exert anticancer effects on human breast cancer MCF-7 cells and human colon adenocarcinoma cells through cell cycle arrest (Corona et al., 2009; Han et al., 2009). Besides, HY could guard against angiostenosis and thrombus by reducing the latelet aggregation and concentration of oxidized low density lipoprotein (LDL) in plasma to prevent cardiovascular and cerebrovascular diseases (Mateos et al., 2016; Leger et al., 2005). In addition, HY could postpone the digestion of starch to alleviate postprandial hyperglycemia through the inhibitory effect on α-glucosidase and decrease serum glucose level in diabetic rats (Hadrich et al., 2015; Jemai et al., 2009). Moreover, HY is an ideal free radical scavenger with a catechol structure. Previous researches have reported that HY possessed strong antioxidant activities in vitro and Rossi and colleagues explained its scavenging effect on superoxide anion from the perspective of chemical bonding (Zou et al., 2012; Guo et al., 2010; Rossi et al., 2017). However, its antioxidant mechanism in vivo is still unclear. Further animal experimentation should be carried out to evaluate the antioxidant effect of HY in vivo.
Caenorhabditis elegans is a simple eukaryotic multicellular organism with small size, transparent body, short life-cycle and rapid fertility. These characteristics make it good for breeding and observation. And the complete genome of C. elegans have already been sequenced which was highly homologous to human genes (Stein et al., 2001). More importantly, construction of RNAi library and the application of transgenic strains, especially those coupled with GFP (green fluorescent protein) gene on related functional genes, made genetic analysis of worms more rapid, sensitive and efficient (Fraser et al., 2000). Based on the multiple advantages, C. elegans was widely applied to screen the active components from natural sources. Zhao and co-workers used C. elegans to evaluate the bioactivity of the hydrolysate derived from Strongylocentrotus nudus and found the hydrolysis product could reduce the ROS level and the expression of superoxide dismutase (SOD-3) as well as heat shock protein (HSP-16.2) in C. elegans under oxidative stress (Zhao et al., 2018). And the proteolytic peptides from Angelica sinensis could also played the same role in C. elegans (Qiang et al., 2016). Xu and colleagues used C. elegans as a model to measure the antioxidant activity of polysaccharide from Epimedium acuminatum and found that the polysaccharide could inhibit lipid peroxidation and protein carbonylation in worms, and increase the activities of SOD, catalase enzyme (CAT) and glutathione peroxidase (GSH-Px) (Xu et al., 2016).
Although HY was proved to show an excellent antioxidant ability in vitro, the antioxidant capacity in vivo is still needed further investigation to reveal. Therefore, in this study, C. elegans was used as a model organism to evaluate the in vivo antioxidant activity and explore the mechanism. The results were expected to provide a theoretical basis for the application of HY as well as olive leaves in functional foods, cosmetics and medicine, providing technical support for improving the profitability of olive groves.
2 Materials and methods
2.1 Materials and chemicals
HY was purchased from Shanxi Mirkang Biotechnology Co., Ltd. (Xi’an China).
2,2-Diphenyl-1-picryl-hydrazyl (DPPH) was purchased from Sigma Chemical Co. (USA). Reactive Oxygen Species Assay Kit (ROS) and total RNA kit were purchased from Beyotime Biotechnology (Shanghai, China). Malondialdehyde (MDA) assay kit (TBA method) and the total protein assay kit (with standard: BCA method) were bought from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). fetal bovine serum (FBS) was obtained from Gibco. Dulbecco's modified eagle medium (DMEM), phosphate buffer solution (PBS) and digestive enzyme were purchased from Hyclone (USA). All chemicals were analytical grade. Peptone, tryptone and yeast extract were obtained from OXOID Co., Ltd. Prime Script RT kit and SYBR Premix Ex TaqII kit were bought from Takara Biomedical Technology Co., Ltd. (Beijing, China).
2.2 Determination of antioxidant activity of HY in vitro
2.2.1 Preparation of HY
1 g of HY accurately weighed was dissolved in 100 ml of distilled water, and then filtered through a 0.22 μm microporous nylon membrane. The mother solution was placed in a 4 °C refrigerator for later assays.
2.2.2 Scavenging ability on DPPH free radical
The DPPH free radical scavenging ability of HY was conducted according to the method with some modifications (Brand et al., 1995). Briefly, 35 μL different concentration of HY solution was added with 165 μL of DPPH ethanol solution for 10 min at dark. Ascorbic acid (Vc) was used as the positive control. The absorbance of the mixture was measured at 517 nm and the clearance rate was calculated according to the following formula:
Where the A0 was the absorbance of the blank control group with distilled water instead and A1 was the absorbance of trial group with sample solution.
2.2.3 Determination of reducing power
The total reducing power of HY was determined according to the previous method (Luo et al., 2019). 0.2 ml of HY sample solution (Vc was used as positive control group) was mixed with 0.5 ml phosphate buffer (0.2 M) and 0.5 ml K3[Fe(CN6)] (1% w/v), then the mixture was placed at 50 °C for 20 min. Subsequently the reaction solution was cooled at 0 °C for 5 min before adding 0.5 ml trichloroacetic acid (10% w/v). Then centrifuged and 0.5 ml aliquot of supernatant fluid was added into a tube with 0.1 ml FeCl3 (0.1% w/v) and 0.5 ml distilled water. The absorbance of the reaction solution was read at 700 nm by a microplate reader. The results were expressed as the actual absorbance. An increasing absorbance indicated an ascending reducing power.
2.2.4 Cell culture
Chinese hamster ovary cells (CHO cells) were obtained from the Biotechnology Center of Sichuan University (Sichuan, China). CHO cells were cultured in DMEM containing 10% FBS at 37 °C and 5% CO2 saturation humidity. When the cell density reached about 70%, the cells was digested from the flask and washed twice by PBS. Then the cells were centrifuged at 1000 r/min for 3 min and suspended with fresh medium for following assays.
2.2.5 Establishment of oxidative damage model
When the cells were in log phase, digestion enzyme was used to remove the cells from the bottle and the cells were washed with PBS. The cell suspension was adjusted to the density of 105 cells/ml using ahemocytometer. 90 μL of cell suspension was added to a 96-well plate for 12 h. Then 10 μL different concentrations (0–2 mM) of H2O2 was transferred into each well for 5 h. After incubation, new fresh medium containing 10% CCK-8 was added for 30 min at 37 °C after all the solution was removed. The absorbance of each well was measured at 450 nm. The cell viability was calculated according to the formula:
Where the A0 was the absorbance of blank control group and A1 was the absorbance of the trial group containing sample solution.
2.2.6 Cytotoxicity test of HY
HY was dissolved in DMEM and filtered through a 0.22 μm microporous nylon membrane. The cell suspension was planted in the plate for 12 h and the different concentration of HY solution was added into each well for 48 h at 37 °C. Then fresh medium containing 10% CCK-8 was added after the old solution was removed. The absorbance of the plate was read at 450 nm after 30 min incubation. The cell viability was calculated according to formula (2).
2.2.7 Protection of HY on CHO cells
Cell suspension was adjusted to 105 cells/ml and 90 μL suspension was added into a 96-well plate. The HY sample solution was transferred into the plate for 48 h after cells was incubated for 12 h. The blank control group was added into PBS instead. After the treatment, all solution was removed and PBS was added to wash the cell for twice and then H2O2 was added into each well to create an oxidative stress for 5 h. Subsequently, all solution was removed and the cell was washed with PBS to remove all the H2O2. Then new medium containing 10% CCK-8 kit was added for 30 min. The absorbance of the plated was measured at 450 nm and the cell viability was calculated according to formula (2).
2.2.8 Determination of the ROS level
CHO cell suspension was adjusted to the concentration of 106 cells/ml. 1.8 ml cell suspension was planted into a 6-well plate for 12 h incubation. 0.2 ml HY solution was added to treat the cell for 48 h. Then H2O2 was transferred into the plate for 5 h after all the solution was removed and the cell was washed with PBS. Subsequently, the old solution was removed and the cell was washed twice with PBS. Then ROS detection reagent was added into each well for 30 min. The fluorescence intensity of the plate was read through a microplate reader.
2.2.9 Determination of MDA content
The cell was seeded in a 6-well plate for 12 h. Then HY was added to treat the cell for 48 h·H2O2 solution was transferred into each well for 5 h after the solution was removed completely. The cells were then digested from the flask into a tube. The cells were disrupted by an ultrasonic homogenizer (Scientz-IID, Ningbo, China) and the protein and the MDA content in the supernatant was measured according to the instruction of kit.
2.3 Determination of antioxidant activity of HY in vivo
2.3.1 Cultivation of C. Elegans
The Wide-type Bristol N2, transgenic nematode strains and Escherichia coli OP50 were provided by Caenorhabditis Genetics Center (CGC). The transgenic nematode stains used in this work were as follows: TJ356, zIs356 [daf-16p::daf-16a/b::GFP + rol-6(su1006)]; CF1553, muIs84 [(pAD76) sod-3p::GFP + rol-6(su1006)]; TJ375 (gpls[hsp16.2::GFP]). All strains and OP50 were cultured at 20 °C. C. elegans was maintained on a solid medium plate named nematode growth medium (NGM) with a layer of OP50. The NGM mixed with or without HY before pouring the plates and the same treatment of bacteria solution was applied to each NGM. Worms cultured for about 3 days were applied to get synchronized worms by the sodium hypochlorite method (Portadelariva et al., 2012). Age-synchronized eggs were placed on NGM.
2.3.2 Measurement of body length
The synchronized worms were placed on a new NGM containing different concentration of HY. On day 2 and day 5, the worms were fixed in a glass slide by 0.1% NaN3. Then the slide was observed with a fluorescence microscope (Nikon-Eclipse Ci-L, Japan). The body length of the worms was measured by Image J. Fifteen worms were randomly picked from each treatment group, and the test was repeated three times.
2.3.3 Fertility assay
After the sodium hypochlorite treatment, the eggs were transferred to the medium in the absence or presence of HY. After the eggs were hatched, a single worm was transferred to a 35 mm culture plate. The worms were transferred on a new NGM every 24 h during the egg stage. The transferred plates were kept in the incubator and incubated at 20 °C for 72 h. The number of offspring was observed and recorded under a stereomicroscope. Each group was 10 worms in a parallel, and the test was repeated three times.
2.3.4 Measurement of pharyngeal pumping and body bending frequency
The motor ability on different stages of N2 worms development was judged by the pharyngeal pumping and the body bending frequency, which were performed at day 2 and 5 respectively. The pharynx was pumped up and down once for a pharyngeal pumping movement; the midsection of the worm was bent left and right once for a body bending. the number of pharyngeal pumping was observed and recorded for 30 s under a stereomicroscope. The bending times was observed within 30 s after the worms adapt to the new environment for 2 min on a new plate. 15 worms were in each group, and the test was repeated three times.
2.3.5 Thermal stress
The eggs were placed on the NGM plate with HY for 3 days. After treatment, the worms were transferred on a new plate sealed with parafilm under 35 °C for 5 h of acute heat stress treatment, and the survival numbers were recorded. The death of worm was identified by touching the head and tail without any response. The worms climbing to the wall of the petri dish and dehydrating were recorded as escape. There were 30 worms in each group, and the test was repeated three times.
2.3.6 Determination of ROS
The N2 worms pretreated with HY for 3 days were placed at 35 °C for 1 h. Then the worms were washed from NGM with M9 kept in a 35 °C water bath and the worms were repeatedly washed 3 ∼ 6 times to remove OP50. After centrifugation, the worms were mixed with 1 ml of 100 μmol DCFH-DA reactive oxygen test reagent, and the mixture was incubated for 30 min in the dark on a horizontal shaker. The fluorescence was measured by a fluorescence microplate reader with the excitation wavelength 488 nm and emission wavelength 525 nm. The number of worms per experiment was about 30 and 3 independent experiments were performed.
2.3.7 Determination of MDA content and antioxidant enzyme activities
The N2 worms was treated with HY for 3 days and then the worms were undergone heat stress at 35 °C for 1 h. The worms were washed from NGM with M9, and repeatedly washed 3 ∼ 6 times with M9 kept in a 35 °C water bath until the worm solution was clear and transparent. Then the worms were crushed by an ultrasonic wave to obtain the homogenate. The supernatant after centrifuged at 10,000 r/min for 10 min was used to determine the protein content, MDA content and the abilities of antioxidant enzymes according to the instructions of BCA, MDA, SOD and GSH-Px kit. The number of worms per experiment was about 2000 and 3 independent experiments were performed.
2.3.8 Visualization of the HSP-16.2::GFP
The TJ375 worms were treated with HY for 3 days before placed at 35 °C for 1 h. Then the worms were washed from NGM with M9 buffer and the worms were repeatedly washed 3 ∼ 6 times to remove all OP50 strain. The worms was anesthetized by adding a 0.1% NaN3 solution and fixed on a glass slide to observe the expression of HSP-16.2::GFP (Nikon-Eclipse Ci-L, Japan) and the fluorescence density was analysized by Image J software. The number of worms per experiment was 15 and 3 independent experiments were performed.
2.3.9 Visualization of the SOD-3::GFP
The CF1553 worms were maintained on the NGM plate with HY for 3 days before the plate was placed at 35 °C for 1 h. Then the worms were washed from NGM with M9 buffer and repeatedly washed 3 ∼ 6 times to remove all OP50 strain. The worms were anesthetized by adding a 0.1% NaN3 solution and fixed on a glass slide to observe the expression of SOD-3::GFP by a fluorescence microscopy (Nikon-Eclipse Ci-L, Japan) and the fluorescence density was analysized by Image J software. The number of worms per experiment was 15 and 3 independent experiments were performed.
2.3.10 Nuclear localization of DAF-16
Carrying a DAF-16::GFP, the TJ356 worms were applied to observe the translocation of DAF-16 in the nucleus. Briefly, synchronized eggs were transferred to a fresh NGM plate with the absence or presence of the HY for 3 days. Then the worms were obtained from the plate and washed with M9 for 3 ∼ 6 times until all the OP50 strain was removed. The worms were transferred on a glass slide with 0.1% NaN3 solution to be anesthetized. Then the subcellular distribution of DAF-16::GFP was observed under a fluorescence microscope (Nikon-Eclipse Ci-L, Japan). The location of DAF-16 was categorized as cytosolic, intermediate and nuclear. The number of worms per experiment was 15 and 3 independent experiments were performed.
2.3.11 The expression of relative genes by qPCR
N2 worms pretreated with HY for 3 days was washed with M9. Then the worms was frozen with liquid nitrogen and ground instantly. The total RNA was extracted with a simple Total RNA kit and the quality of the RNA was evaluated using NanoDrop®2000 and agarose gel electrophoresis. According to the instructions of the Prime Script RT kit, RNA was reverse transcribed into cDNA immediately and the cDNA was stored in a refrigerator at −20 °C. According to the instructions of the SYBR Premix Ex TaqII kit, the qPCR program was performed on CFXTM96 Real-time system (BIO-RAD, California, USA) with following protocol: 95 °C for 3 min; 95 °C for 10 s, 60 °C for 30 s, 40 cycles and collected the fluorescence signal; rise 0.5 °C every 5 s from 65 °C to 95 °C, kept for 5 s, and collected the fluorescence signal separately. Act-1 was used as the internal reference gene. The primer sequences were shown in Table 1. The relative expression of antioxidant-related genes in C. elegans was analyzed using 2−ΔΔCT method (Livak and Schmittgen, 2001). The number of worms per experiment was about 2000 and 3 independent experiments were performed.
Genes
Primers
act-1
Forward
5‘-CCAGGAATTGCTGATCGTATGCAGAA-3′
Reverse
5‘-TGGAGAGGGAAGCGAGGATAGA-3′
hsp-16.2
Forward
5‘-CTGCAGAATCTCTCCATCTGAGTC-3′
Reverse
5‘-AGATTCGAAGCAACTGCACC-3′
ctl-2
Forward
5‘-TTCGCTGAGGTTGAACAATCCG-3′
Reverse
5‘-GTTGCTGATTGTCATAAGCCATTGC-3′
sod-3
Forward
5‘-ATTCGCCAACCCATGATGG-3′
Reverse
5‘-GCTCCCAAACGTCAATTCCA-3′
daf-16
Forward
5‘-TTTCCGTCCCCGAACTCA-3′
Reverse
5‘-ATTCGCCAACCCATGATGG-3′
age-1
Forward
5‘-CCTGAACCGACTGCCAATC-3′
Reverse
5‘-GTGCTTGACGAGATATGTGTATTG-3′
daf-2
Forward
5‘-GGATAAAGGCGAATCAAAGTGTC-3′
Reverse
5‘-CGATACACTTTCCCTTGTGATAGAC-3′
2.4 Statistical analyses
The data in this study were analyzed statistically by Student’s t-test (t-test) and ANOVA (GraphPad Prism 6) (GraphPad Software, Inc., La Jolla, CA, USA). The fluorescence intensity was quantified by the Image-J software (National Institutes of Health, Bethesda, MD, USA) All experiments were performed in triplicates and the results were expressed as mean ± standard deviation (SD), n = 3. Difference at the p < 0.05 level was considered to be significantly different.
3 Results
3.1 HY possessed a strong antioxidant ability in vitro
DPPH radical ethanol solution had a strong absorption peak at 517 nm. The absorbance value can be detected for assessing the antioxidant ability (Mishra et al., 2012). As shown in Fig. 1A, HY had a strong ability to clear DPPH with an maximum clearance of 85.65% at the concentration of 1.0 mg/ml. When the concentration reached 0.4 mg/ml, the clearance rate tended to be flat. The reducing power was associated with the antioxidant ability to some degree. [Fe2+(CN)6]4- had a characteristic absorption peak at 700 nm and antioxidants could change [Fe3+(CN)6]3- to [Fe2+(CN)6]4- to increase the absorbance. Hence the reducing power was positively correlated with the absorbance (Benzie et al., 1996). As shown in Fig. 1B, the total reducing power of HY was promoting with the increasing concentrations, showing a good dose–effect relationship.
(A) The DPPH free radicals scavenging activity and (B) the total reducing power of HY.
3.2 The antioxidant of HY on CHO cells
3.2.1 Injury model
CHO cells were treated with different concentrations of H2O2 (0–2 mM) for 5 h and the cell growth was inhibited to some degree. Compared with the blank control group, the cell viability was reducing with the increasing of H2O2 (Fig. 2A). Among them, 1.5 mM H2O2 reduced the cell viability to about 50% of normal cells. At this concentration, CHO cells both had oxidative damage and retained half of their activity. Therefore, 1.5 mM H2O2 was selected to establish the CHO cell oxidative damage model (Xia et al. 2017).
The protection of HY on CHO cells under oxidative stress. (A) the cell viability of CHO cell exposed to different concentration of H2O2 and the cell viability of CHO cells under different concentration of HY. (C)After pretreated with HY, the cell viability of CHO cell under the oxidative stress. The significance was evaluated by ANOVN compared with the cell viability of H2O2 group. * meant p < 0.05 and *** meant p < 0.001.
3.2.2 HY protected CHO against H2O2
When the drug concentration was too high, the cell viability would be reduced by affecting the osmotic pressure of the cell. Therefore, it was necessary to screen the non-toxic HY concentration first. 20–100 μg/ml of HY showed no toxic effect on CHO cells, and cell viability was equivalent to that of the blank control group. While HY was 200 μg/ml, the cell viability decreased by 40.05%, showing a toxic effect (Fig. 2B). Therefore, the concentration of HY in subsequent experiments was controlled within 100 μg/ml. As showing in Fig. 2C, pretreated with HY for 48 h, the cell viability was enhanced compared with the injury group showing the significant protective effect of HY on CHO cells against oxidative damage. And the impact was gradually enhanced in the test concentration. Hence, in the following assays, 80 and 100 μg/ml of HY was chosen for the suitable concentration.
3.2.3 HY reduced ROS and MDA in CHO cells
H2O2 stimulation increased the ROS level in the model group. As shown in Fig. 3A, compared with the injury group, 80 μg/ml of HY could reduce significantly the ROS level by 10.28% and 100 μg/ml of HY could reduce 15.62%. Lipid peroxidation could lead to membrane lipid oxidation, which was the main component of cell membranes, causing cell death. The final product of lipid peroxidation was malonaldehyde (MDA). H2O2 stimulation could increase the content of MDA (Fig. 3B) in injury group while the content in the cells pretreated with HY for 48 h was significantly decreased, which might because HY could reduce the increasing MDA caused by exogenous oxidative stimulation. The lower of ROS and MDA may be related to the activation of intracellular antioxidant defense mechanism by HY (Hu et al., 2017).
Under the oxidative stress, after pretreated with HY, (A) the ROS level and (B) the MDA content in CHO cells. The significance was evaluated by ANOVN compared with the cell viability of H2O2 group. * meant p < 0.05.
3.3 The antioxidant potential of HY on C. Elegans
3.3.1 HY showed no toxic effect on development
The effects on physiological activities were usually used to evaluate the toxicity of drugs, such as development and fertility. Body length is an important indicator for assessing development. As shown in Fig. 4A, compared with the blank control group, with treatment of HY at different concentration, the length of the worms was slightly increased or inhibited, but there was no statistical difference, indicating that HY at the test concentration showed no toxic effect on the worms. And compared with the reported literature (McCulloch and Gems, 2003), the body length of worms was within the normal range.
HY showed no toxicity on C. elegans. (A) the body length of C. elegans treated with HY. Fifteen worms were randomly picked from each treatment group, and the test was repeated three times. (B) the effect of HY on the fertility of C. elegans. Each group was 10 worms in a parallel, and the test was repeated three times. The effect of HY on the motility of C. elegans including (C) pharyngeal pumping and (B) body bending. 15 worms were in each group, and the test was repeated three times. The significance was evaluated by ANOVN compared with the blank control. * means p < 0.05 and *** meant p < 0.001. The concentration of EGCG was 50 μg/ml.
3.3.2 HY showed no toxic effect on the fertility of C. Elegans
In order to assess the influence of HY on the reproductive capacity, the number of progenies of the worms was measured. As shown in Fig. 4B, the egg stage of C. elegans started from day 3, both day 4 and day 5 were the peak spawning days, and then the laying gradually decreased until the spawning time ended. The daily spawning amount and the total number of offspring in the HY-treated group were not significantly different from those in the blank control group, indicating that HY had no effect on its fertility cycle and total fertility. The total number of progenies of each treatment group was about 270, which was in line with the previous research (Lewis et al., 1995).
3.3.3 HY showed no toxic effect on the movement of C. Elegans
Pharyngeal pumping and body bending were regulated by complex neural networks and muscle groups, and abnormal motor states could reflect neurological or muscular function defects (Chow et al., 2006; Zhen et al., 2015; White et al., 1986). As shown in Fig. 4C and 4D, there were no statistical differences between the two indicators of average pharyngeal pumping times and body bending times compared with the control group, indicating that HY treatment had no effect on nematode exercise capacity.
3.3.4 HY enhanced the thermal resistance in C. Elegans
Heat stress promoted the body to produce a large number of free radicals forming an endogenous oxidative stress. Testing the protective effect of HY on worms under heat stress could effectively evaluate its antioxidant effect (Alemu et al., 2018). The mortality rate under acute heat stress was an intuitive indicator for evaluating the resistance of HY to the heat shock environment of worms. Compared with the blank group, the survival rate of worms in 50 μg/ml HY group increased by 15.79% and the rate of the 100 μg/ml treatment group was also comparable to the positive control group, reaching 5.93% (Fig. 5A). While the protection ability of HY at 10 μg/ml was relatively weak. The survival rate was only improved by 1.57%, which showed no significant difference with the blank group. In general, HY can significantly improve the heat-resistance of C. elegans. Apart from determining the survival, further investigation would be carried out to explore the mechanism.
The protection of HY on the C. elegans under oxidative stress. (A) the survival of worms exposed to hot stress and there were 30 worms in each group, and the test was repeated three times.. The effect of HY on (B) the ROS level and (C) the content of MDA in C. elegans under stress conditon and the number of worms per experiment was about 2000 and 3 independent experiments were performed. The significance was evaluated by ANOVN compared with the blank control. * means p < 0.05, ** means p < 0.01 and *** meant p < 0.001. The concentration of EGCG was 50 μg/ml.
3.3.5 HY lowered the ROS level in C. Elegans
Excessive accumulation of ROS could damage intracellular DNA, proteins and other biological macromolecules, leading to cell death in severe cases (Toren et al., 2000). So we determined the effect of HY on the ROS level in C. elegans under heat stress. After 1 h heat treatment with 35℃, ROS was sharply accumulated in the worms (Fig. 5B). With the treatment of different concentration of HY, the ROS content was reduced by 8.30, 21.72, 21.79%, respectively, and the positive drug epigallocatechin gallate (EGCG) could reduce the level by 23.85%. Excessive ROS may cause oxidative damage and increase the mortality of worms (Wu et al., 2013). HY could reduce the level of ROS in worms, which was consistent with the results of reducing the mortality.
3.3.6 HY lowered the content of MDA in C. Elegans
As a marker of lipid peroxidation, the content of MDA in worms could reflect the degree of oxidative damage (Esterbauer et al., 1990). 10 μg/ml of HY could significantly reduce the content of MDA with the same effect as the positive control (Fig. 5C). Higher concentration of HY treatment groups could even more reduce the MDA content, lowering lipid peroxidation and alleviating oxidative damage caused by heat stress. The above test results demonstrated that HY could effectively reduce the ROS and MDA levels in C. elegans under heat shock condition, significantly improving the survival rate and the antioxidant effect of HY at 50 μg/ml exhibited the best effect. Therefore, in the following experiments, 50 μg/ml HY was used to explore the mechanism.
3.3.7 HY enhanced the expression of HSP-16.2
Heat shock protein had the function of molecular chaperone. Under environmental stress, the HSP-16.2 could help other proteins fold correctly, keep their original spatial conformation and biological activity, reduce the denaturation and inactivation, and enhance the stress resistance. Lee and co-works found that increasing the expression of HSP-16.2 in worms could reduce oxidative damage and improve the survival under heat stress (Lee et al., 2017). The heat shock protein HSP-16.2 in TJ375 worms was coupled with green fluorescent protein, and its expression could be judged by fluorescence intensity analysis. The fluorescence intensity of HSP-16.2::GFP in blank control was lower (Fig. 6A), while in the HY treatment group the intensity was pretty high (Fig. 6B). After treatment with HY, the expression level of HSP-16.2 in C. elegans increased by 49.01% (Fig. 6C). Hence HY might promote the expression of HSP-16.2 to enhance the thermal stress resistance in C. elegans.
The expression of HSP-16.2::GFP (A) in blank group and (B) in HY treatment group and (C) the effect of HY on the expression of HSP-16.2 under heat stress. The number of worms per experiment was 15 and 3 independent experiments were performed. The effect of HY on the antioxidant enzymes in C. elegans. The expression of SOD-3::GFP (D) in blank group and (E) in HY treatment group and (F) the effect of HY on the expression of SOD-3 under heat stress. The number of worms per experiment was 15 and 3 independent experiments were performed. The activities of antioxidant enzymes in C. elegans including (G) SOD and (H) GHS-Px under hot stress. the number of worms per experiment was about 2000 and 3 independent experiments were performed. The significance was evaluated by t-test. * means p < 0.05, ** means p < 0.01 and *** meant p < 0.001. And the scale bar for A and B micrographs was 50 μm and 100 μm for D and E micrographs.
3.3.8 HY could promote the abilities of antioxidant enzymes
HY increased the resistance of C. elegans to heat stress, which may be related to the regulation of the antioxidant defense system in C. elegans. Therefore, the effects of HY on the antioxidant enzymes were carried out. Based on the measurement of the activity of SOD as well as GSH-Px and the fluorescence intensity of SOD-3::GFP, the results showed that HY could improve the abilities of SOD and GSH-Px by 42.86% and 35.86%, respectively (Fig. 6G and H). HY also significantly improve the fluorescence dendity in CF1553 (Fig. 6D and 6E) and HY increased the expression of SOD-3::GFP (Fig. 6F), indicating that HY may activate the antioxidant enzyme system of C. elegans to quickly respond to excess ROS produced by heat stress, thereby alleviating stress damage and improving survival (Feng et al., 2018).
3.3.9 Effect of HY on nuclear translocation of DAF-16
The transcription factor DAF-16 in C. elegans was the regulating center of multiple signaling pathways and involved in the regulation of important processes such as metabolism, stress response and life cycle (Lee et al., 2003; Ogg et al., 1997). The expression of DAF-16 target genes such as sod-3 and hsp-16.2 was affected by the degree of DAF-16 nuclear localization (Saltiel et al., 2001; Kailiang et al., 2004). HY could up-regulate the expression of hsp-16.2 and sod-3, thus enhancing the stress-resist ability of C. elegans. In order to investigate whether this impact was related to DAF-16, TJ356 strain was applied to explore the effect of HY on DAF-16. The three statuses of the DAF-16 in C. elegans were shown in Fig. 7A (cytosolic), B (intermediate) and C (nuclear). Only in 10.17% of worms, the DAF-16::GFP protein was in the nuclear localization in the blank control group, which was consistent with the previous conclusion under normal growth conditions (Lin et al., 2018). Pretreated with HY, the subcellular localization of DAF-16 changed significantly, and the fusion protein in the nuclear localization increased to 76.13% (Fig. 7D), indicating HY may directly promote the transportation to the nucleus of DAF-16 or act on genes located upstream of it.
Effect of HY on DAF-16 transportation to the nucleus. (A) the cytosolic localization of DAF-16::GFP. (B) both cytosolic and nuclear localization of DAF-16::GFP. (C) the nuclear localization of DAF-16::GFP. (D) the effect of HY on the subcellular localization of DAF-16::GFP. The significance was evaluated by t-test. *** meant p < 0.001. The number of worms per experiment was 15 and 3 independent experiments were performed. And the scale bar for each micrograph was 100 μm.
3.3.10 Effect of HY on the expression of related genes
Based on the results of the above tests, it was inferred that HY could promote the nuclear localization of DAF-16 and up-regulate the expression of sod-3 and hsp-16.2, thus eliminating free radicals, reducing peroxidation products and reducing oxidative damage. In order to further verify it from the mRNA level, the relative expression of related genes was quantitatively determined by qPCR. Daf-16 was a transcription factor of the insulin signaling (IIS) pathway. The IIS pathway played an important role in the regulation of worm stress and lifespan and widely existed in the regulation of life activities of fruit flies, mice, and humans (Murphy et al., 2013). Therefore, in addition to daf-16 and its target genes hsp-16.2 and sod-3, the upstream genes daf-2 and age-1 in this pathway were also examined. The results were shown in Fig. 8. HY could down-regulate the expression of daf-2 and age-1, and up-regulate the expression of daf-16 and sod-3. However, the expression of hsp-16.2 was lower than that of the blank group, which might be probably because the expression of hsp-16.2 would be up-regulated under stress conditions, while the worms used for qPCR were under normal culture conditions. And maybe also HY affected HSP-16.2 in post-transcriptional regulation as well as translation and post-translational regulation (Erin et al., 2019).
The effect of HY on the expression of related genes in C. elegans (A) age-1, (B) daf-2, (C) clt-2, (D) sod-3 and (E) daf-16. The number of worms per experiment was about 2000 and 3 independent experiments were performed. The significance was evaluated by t-test. * means p < 0.05 and *** meant p < 0.001.
4 Discussion
Free radicals participated in many metabolic processes in the body and played an important role in maintaining the body's normal metabolism. However, due to the strong oxidizing capacity, an excess of free radicals would attack the macromolecules such as nucleic acids and proteins and cause oxidative damage. In general, the generation and removal of free radicals were in a balance. But under stress or pathological conditions, the amount of free radicals was often too much to cause damage to lipid, DNA and protein, which required the addition of extra antioxidants or other antioxidant-related factor activators to help maintain oxygen metabolism balance in the body. The DPPH clearance rate of HY was up to 84.81% and the reducing power was comparable to Vc showing a strong antioxidant activity. It probably worked through reacting with free radicals to form stable compounds or directly reducing oxidation products (Huang et al., 2005). Moreover, HY could play an antioxidant role in a variety of ways including chelating metal ions to reduce fenton reaction, acting on free radical-related enzymes—inhibiting the activity of enzymes that caused free radicals and enhancing the activity of free radical scavenging enzymes (Driss et al., 2010; Pazos et al., 2008).
In order to investigate the antioxidant activity of HY on cells, 1.5 mM of H2O2 was used to establish the injury model. Under oxidative stress, cell viability was sharply reduced, and the ROS level as well as MDA content increased, indicating the model was successfully carried out (Shi et al., 2020). Treatment with HY could alleviate oxidative stress in CHO cells, reduce ROS and MDA levels, and enhance cell activity, which was in accordance with previous reports of HY activity on BME-UV1 cells (Basiricò et al., 2019).
Antioxidant activity of HY in vitro was verified in cells. Furtherly, the capacity in vivo was explored by applying C. elegans. Drug treatment could increase the stress resistance of C. elegans, but probably cause toxic and side effects such as retarding development and reducing fecundity (Liao et al., 2011; Peixoto et al., 2019). In order to evaluate the toxicity of HY, the body length, the fecundity, the pharyngeal pumping and the body bending rate of worms were measured. There were no significant differences between trial groups and the blank control group, indicating HY exhibited non-toxic effect on the development, fertility, and movement of C. elegans. All indexes were within the normal range compared with previous reports (McCulloch and Gems, 2003). And the effect on worms motion of HY validated the previous study again that HY have no effect on activity index of young nematodes (Rosa et al., 2020; Brunetti et al., 2020).
50 μg/ml HY could increase the survival rate of worms by 15.79% under thermal stress. Previous reports proved that 400 μg/ml olive leaves extract could also increase the survival by 10.43% (Luo et al., 2019). The concentration of HY used in this study was lower, indicating that HY might be the main active substance in olive leaves extract. Moreover, HY was found to reduce the level of ROS and MDA under heat stress and enhance the expression of heat shock protein HSP-16.2. This indicated that treatment with HY could avoid the damage of intracellular DNA, proteins and other biological macromolecules caused by excessive ROS, reduce lipid peroxidation and alleviate oxidative damage, thus improving the survival rate. Further investigations about antioxidant enzyme, subcellular localization and gene expression were carried out to explore the underlying mechanism. Under favorable conditions in C. elegans, the kinase domain of DAF-2 is activated when insulin-like peptides (ILPs) bind to the receptor. Then activated DAF-2 phosphorylates AGE-1, a phosphatidylinositol 3-kinase to activate downstream kinases, including PDK-1, SGK-1, AKT-1, and AKT-2 protein kinase B proteins. These protein kinases ultimately cause the activity of DAF-16 phosphorylate. Phosphorylation of DAF-16 renders it inactive due to sequestration in the cytoplasm. However, under weakening IIS signal, more nonphosphorylated DAF-16 is targeted to the nucleus, where it controls the expression of genes involved in metabolism, immune defense, autophagy, and stress resistance (McCulloch and Gems, 2003; Lin et al., 2001; Taguchi and White, 2008). The results showed HY could promote the nuclear localization of DAF-16 and increase the content of SOD-3::GFP as well as the activities of SOD and GSH-Px. Moreover, the qPCR results showed HY could reduce the expression of upstream component daf-2 and age-1, increase the expression of daf-16 and sod-3. Previous research has suggested that the decrease of daf-2 and age-1 could reduce the phosphorylation of DAF-16 and promote its transportation to the nucleus (Lu et al., 2017), and also probably reduce the inhibition of downstream component daf-16 (Wang et al., 2018), so that the expression of daf-16 and its target gene sod-3 were up-regulated. It was suggested that HY could alleviate the oxidative damage by affecting the IIS signaling pathway in C. elegans. And in previous studies, Rosa and Brunetti also found the benefit effect of HY on survival enhancement under heat stress and amelioration of Parkinson’s disease (Rosa et al., 2020; Brunetti et al., 2020). In addition, Rossi and colleagues found that extra virgin olive oil which containing HY could significantly increase nematodes life span and survival of paraquat-exposed and SKN-1/Nrf as an important regulator of oxidative stress which may be another target of the protective effects of HY (Rossi et al., 2017; Blackwell et al., 2015). That is to say, the antioxidant capacity of HY was emphasized as the main biochemical mechanisms responsible for the improvement of diverse health attributes. Further biological and medical trials are indicated to assess the full potential of HY and to uncover their mechanism.
5 Conclusions
HY could well scavenge DPPH free radical and possesses a strong total reducing power. Also HY exhibited a good protective effect on oxidative damage of CHO cells by reducing intracellular ROS and MDA content. In vivo assays, C. elegans was used as the model organism. HY could probably inhibit IIS signaling to promote the expression and nuclear translocation of DAF-16, thereby promoting the expression of downstream protective factors to reduce the peroxidation product and enhance the thermal resistance of C. elegans without toxicity. In summary, HY showed a strong antioxidant ability in vitro and in vivo.
Author Contributions
All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the Sichuan Science and Technology Program, Sichuan, China, grant number 2020YFH0207.
CRediT authorship contribution statement
Yujie Wang: Investigation, Writing - original draft. Siyuan Luo: Investigation. Zhou Xu: Resources. Li Liu: Resources. Shiling Feng: Software. Tao Chen: Formal analysis. Lijun Zhou: Formal analysis. Ming Yuan: Methodology. Yan Huang: Methodology. Chun bang Ding: Writing - review & editing.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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