Abstract : Objective To investigate the protective effect of water-soluble coenzyme Q10 (CoQ10) and its possible mechanism in the Rot-induced Parkinson's disease (PD) cell model, and to provide a theoretical basis for the use of CoQ10 in the treatment of PD. Methods PC12 cells in logarithmic growth phase were divided into control group, Rot group, CoQ10 group and CoQ10 treatment group; cell viability was detected by CCK8; intracellular reactive oxygen species (ROS) level was measured by fluorescence spectrophotometer using fluorescent probe 2 ′,7 ′-dihydrodichlorofluorescent yellow bis-acetate (DCFH-DA); and the effect of CoQ10 on the apoptosis signaling protein Bcl-2,3-dihydrodichlorofluorescent yellow bis-acetate (Bcl-2,4-dihydrodichlorofluorescent yellow bis-acetate) was detected by Western blot. The effects of CoQ10 on the expression of apoptosis signaling proteins Bcl-2, Bax, Cas pas e-9, active Cas pas e-3 and apoptosis-inducing factor (AIF) were detected by Western blot.
The survival rate of PC12 cells after 24 h of Rot treatment was significantly decreased (P＜0.01) and was negatively correlated with the dose, and the level of ROS was increased by Rot (P＜0.01). 01) and was negatively correlated with the dose, and Rot induced an increase in the ROS level (P＜0.01), CoQ10 improved the survival rate of PC12 cells induced by Rot treatment. 01), CoQ10 could improve the Rot treatment-induced decrease in PC12 cell survival (P＜0.01) and decrease ROS level (P＜0.01). CoQ10 improved the Rot treatment-induced decrease in PC12 cell viability (P<0.01) and ROS level (P<0.01); Western blotting improved PC12 cell viability (P<0.01) and ROS level (P<0.01). Western blot showed that CoQ10 reduced Rot-induced Cas pas e-9 (P<0.05), active Cas pas e-9 (P<0.05), and active Cas pas e-9 (P<0.05). Western blot showed that CoQ10 reduced Rot-induced Cas pas e-9 (P<0.05), active Cas pas e-3 (P<0.05) and Bax (P<0.05). (P<0.05), active Cas pas e-3 (P<0.05) and Bax (P<0.01). (P<0.05), active Cas pas e-3 (P<0.05), and Bax (P<0.01), and up-regulated Bcl-2 (P<0.01). (P<0.01); prevented AIF from translocating to the nucleus (P<0.05). (P < 0 .05) .
Conclusion: Water-soluble CoQ10 has a protective effect against apoptosis in Rot PC12 cells, which may be achieved by removing intracellular oxygen radicals to improve the cellular oxygenation stress state, blocking the translocation of AIF to the nucleus, up-regulating Bcl-2, down-regulating Bax, and decreasing the expression of active Cas pas e-3 and Cas pas e-9.
Parkinson's disease (PD), also known as paralysis agitans, is a neurodegenerative disease common in the elderly, clinically characterized by resting tremor, bradykinesia, myotonia and postural balance disorders. A combination of genetic and environmental factors plays an important role in the development of PD, and the main pathologic change is degeneration of nigrostriatal dopaminergic neurons. Although the exact pathogenesis of PD has not yet been clarified, a series of studies have shown that the pathogenesis of PD is mainly related to activation of the apoptotic pathway, oxidative stress and mitochondrial dysfunction[1-2] .
Coenzyme Q10 (CoQ10) acts as an uncoupling protein for mobile electron transport and cofactors in the mitochondrial respiratory chain, and is a powerful antioxidant in the human body, preventing oxidative damage by free radicals, including the oxidation of lipids in the mitochondrial membrane. It has been suggested that the occurrence of PD may be related to the reduction of CoQ10[3-4] . However, traditional CoQ10 is fat-soluble and has a low bioavailability, which is difficult to be absorbed and utilized by the human body. Recently developed water-soluble CoQ10 has a cellular uptake and mitochondrial uptake that are 60 and 20 times higher than that of traditional CoQ10, respectively[5] .
Rotenone (Rot) is a common insecticide that acts in the same way as the pro-parkinsonian toxin 1-methyl-4-phenylpyridine (MPP+), specifically inhibiting NADH dehydrogenase (mitochondrial complex I) of the cellular respiratory chain[6] . The nature, enzymes and transmitters synthesized by rat pheochromo cytoma cell (PC12) are similar to those of midbrain dopamine neurons[7-8] . Therefore, the present group used PC12 cells as a model of dopaminergic neurons to study the inhibitory effect of water-soluble CoQ10 on apoptosis, and to provide a basis for clinical application. Therefore, our group used PC12 cells as a cell model of dopaminergic neurons to study the inhibitory effect of water-soluble CoQ10 on neuronal apoptosis and to provide experimental basis for clinical application.
1 Materials and Methods
1 . 1 Main reagents
DMEM high sugar medium, fetal bovine serum (fe- tal bovine serum), PBS phosphate buffer were purchased from Hyclone; penicillin, streptomycin, trypsin were purchased from Solepol; Rot, high purity (>98%) CoQ10, polyoxyethanyl-α-tocopheryl sebacate (PTS) were purchased from Sigma; reactive oxygen species (ROS) detection kit was purchased from Nanjing Jianjian Institute of Biological Engineering; reactive oxygen species (ROS) detection kit was purchased from Nanjing Jianjian Institute of Biological Engineering; ROS detection kit was purchased from Nanjing Jianjian Institute of Biological Engineering; ROS detection kit was purchased from Nanjing Jianjian Institute of Biological Engineering. Rot, high purity (>98%) CoQ10, polyoxyethanyl-α-tocopheryl sebacate (PTS) were purchased from Sigma; reactive oxygen species (ROS) detection kit was purchased from Nanjing Jianjian Bioengineering Institute; anti-apoptosis-inducing factor antibody (anti-AIF antibody), anti-AIF antibody and anti-AIF antibody were purchased from Nanjing Jianjian Bioengineering Institute; and anti-AIF antibody and anti-AIF antibody were purchased from Nanjing Jianjian Bioengineering Institute. The anti-apoptosis-inducing factor antibody (anti-AIF antibody), anti-active Caspase3 antibody, anti-Caspase9 antibody, anti-beta actin antibody, Anti-Lamin B1 antibody, Goat Anti-Caspase3 antibody, and Anti-Lamin B1 antibody were purchased from Nanjing Jianjian Bioengineering Research Institute. Antibody and Goat Anti-rabbit antibody were purchased from Abcam, USA; plasma protein and nucleoprotein extraction kit was purchased from KGI Biotechnology Co.
1 . 2 Cell lines
Rat adrenal pheochromocytoma PC12 cells (differentiated by nerve growth factor) were purchased from Shanghai Academy of Life Sciences.
1 . 3 Main instruments
Carbon dioxide (CO2) incubator (For-ma Scientific, USA); common inverted microscope (Olympus CKX41, Japan); fluorescence microscope with camera (Nikon eclipse, Japan); fluorescence spectrophotometer (Olympus, Japan); flow cytometer (Olympus, Japan); multiwavelength enzyme assay (Thermo Fisher Scientific, USA). The results were summarized as follows: Fluorescence spectrophotometer (Olympus, Japan); Flow cytometer (Olympus, Japan); Multiwavelength enzyme assay (Thermo, USA).
1 . 4 Methodology
1 . 4 . 1 Reagent preparation
Rot was dissolved in DMSO to make a storage solution and set aside; PTS was mixed with CoQ10 in a 2:1 molar ratio at 50 ℃ to make water-soluble CoQ10[9] .
1 . 4 . 2 Cell Culture
PC12 cells were incubated in DMEM high glucose medium containing 100 mL/L fetal bovine serum, 1% streptomycin, 50 mL/L CO2, and 37 ℃ saturated humidity incubator for 24 h. When the cells were fused to about 80%, they were used for the following experiments.
1 . 4 . 3 Rot Modeling PD
The logarithmic growth phase cells were inoculated into 96-well plates and incubated at 37 ℃ overnight in an incubator with different concentrations of Rot (0.25, 0.5, 0.5, 0.5, 0.5, 0.5, 0.5, 0.5). 25 、0 . 5 、1 . The cells were incubated at 37 ℃ overnight with different concentrations of Rot (0.25, 0.5, 1.0, 2.0). The logarithmic growth phase cells were inoculated into 96-well plates and incubated in a 37℃ incubator for 24 h. Different concentrations of Rot (0.25, 0.5, 1.0, 2.0 μmol/L) were used to act on the cells for 24 h. At the same time, a blank control group and a solvent control (vehicle) group (1 mL/L DMSO) were set up, and the cell viability was detected by using CCK8, and the morphology of the cells was observed under the inverted microscope.
1 . 4 . 4 Effect of water-soluble CoQ10 on the survival of normal cells and PD-model cells
Logarithmic growth phase cells were inoculated into 96-well plates, incubated at 37 ℃ for overnight, pre-treated with different concentrations of water-soluble CoQ10 (25, 50, 100 μmol/L) for 3 h, and then treated with ichthyospermone for 24 h. The survival rate of the cells was examined by CCK8, and the cell morphology was observed in an inverted microscope.
1. 4. 5 Experimental groups
The cells were divided into vehicle group (1 mL/L DMSO), Rot group (1 μmol/L Rot), CoQ10 group (50 μmol/L CoQ10), and CoQ10 treatment group (1 μmol/L Rot + 50 μmol/L CoQ10).
1 . 4 . 6 Intracellular ROS Detection
ROS was detected by using the Nanjing Built ROS Assay Kit. 1 × 106 cells were collected from vehicle group, Rot group, CoQ10 group, CoQ10 treatment group and positive control group (Ros up, 50 mg/L, provided in the kit), and resuspended in PBS containing 10 μmol/L 2′,7′-dihydrodichlorofluorescent yellow bis-acetic acid sodium salt (DCFH-DA), incubated for 30 min in CO2 incubator, washed three times in PBS, and the cell density was adjusted with PBS to make the concentration of cells in each group consistent. Incubate in CO2 incubator for 30 min, wash with PBS for 3 times, resuspend the cells with PBS, and adjust the cell density so that the concentration of cells in each group is the same. The positive control group was incubated with Rosup at a final mass concentration of 50 mg/L for 30 min according to the requirements of the kit, and the fluorescence intensity was detected by a fluorescence spectrophotometer, with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The ratio of each treatment group/control group was used as the value of ROS.
1 . 4 . 7 Effect of CoQ10 on the expression of apoptotic signaling proteins AIF, Bcl-2, Bax, Caspas e-9, and active Caspas e-3 by Western blotting
The processed cells were collected, and plasma proteins and nuclear proteins were extracted according to the requirements of the kit (KGI Nuclear Protein Plasma Protein Extraction Kit), and the protein content was determined by Bradford's method, and the protein content was separated and mirrored by SDS-PAGE (Nikon eclipse, Nikon, Japan), fluorescence spectrophotometer (Olympus, Japan), flow cytometer (Olympus, Japan), and multiwavelength enzyme assay (Thermo, USA). The results were analyzed by a flow cytometer (Olympus, Japan) and a multiwavelength enzyme assay (Thermo Fisher Scientific, U.S.A.).
1.5 Statistical Processing All data were analyzed using Excel as the database and GraphPad Prism 5.0 as the statistical software.
Measurements were expressed as ± s. One-way ANOVA (Newman-Keul s) was used to analyze the two-way comparisons between groups and between groups, and differences were considered statistically significant at P<0.05.
2 Results
2.1 Effect of different concentrations of Rot on the survival rate of PC12 cells In this experiment, cells were treated with gradient concentration of Rot for 24 h. The cell survival rate was shown in Fig. 1. One-way ANOVA showed that there was a statistically significant difference in the cell survival rate between different concentrations of Rot (P＜0.01), and the cell survival rate was negatively correlated with the concentration of Rot, and the higher the concentration of Rot, the lower the cell survival rate was. Under the inverted microscope, the morphology of the cells was observed. In the normal control group and the solvent control group, the cell protrusions were obvious and pike-shaped, but after treatment with different concentrations of Rot, most of the cell bodies were wrinkled, the protrusions were shortened or even disappeared, and the cells were similar to a round shape, and the number of cells was reduced. At a concentration of 1 μmol/L Rot, the cell survival rate was about 50%, and 1 μmol/L Rot will be used to establish the cell model in the subsequent experiments. there was no statistical significance in the difference between the control group and the vehicle group (P＞0.05), and the effect of DMSO on the cells could be ignored.
Fig. 1 Effect of different concentrations of Rot on the viability of PC12 cells.
Fig. 1 Effects of Rot of different concentration on survival rate of PC12 cells
Compared with the Vehicle group,∗ P < 0. 05,∗ ∗ P < 0. 01.
2.2 Effect of water-soluble CoQ10 on the morphology and survival of Rot PC12 cells
PC12 cells in the model group were treated with 25, 50 and 100 μmol/L CoQ10, and the results showed that 50 and 100 μmol/L CoQ10 increased the cell survival rate of the Rot group compared with that of the Rot group (P＜0.05), but there was no significant difference between 50 μmol/L and 100 μmol/L CoQ10 on the survival rate of PC12 cells (Figure 2). However, there was no significant difference between 50 μmol/L and 100 μmol/L CoQ10 on PC12 cell viability (Figure 2). Therefore, 50 μmol/L CoQ10 was chosen as the therapeutic concentration in the subsequent experiments.
2.3 Detection of intracellular ROS level As shown in Figure 3, the fluorescence intensity of Rot-treated cells in the model group increased significantly after 24 h, suggesting that the intracellular ROS level was elevated; the fluorescence intensity of the treatment group weakened after 3 h of CoQ10 pretreatment, suggesting that the intracellular ROS level was reduced (P＜0.01), and the fluorescence intensity of the CoQ10 group was slightly reduced compared with that of the vehicle group, but the difference was not statistically significant (P＞0.05). The fluorescence intensity of the CoQ10 group was slightly lower than that of the vehicle group, but the difference was not statistically significant (P＞0.05).
2.4 Effects of water-soluble CoQ10 on the expression of apoptosis-inducing protein AIF and apoptosis signaling proteins Bcl-2, Bax, Caspase-9 and active-Caspase-3 The expression of apoptosis signaling proteins was also significantly altered, mainly showing a decrease in Bcl-2 expression (P < 0.01), an increase in Bax and Caspas e-9 expression (P < 0.01), and a significant increase in active-Caspas e-3 expression (P < 0.05). Water-soluble CoQ10 reversed the expression of the above apoptotic signaling proteins (Figure 4).
3 Discussion
The pathogenesis of PD is still controversial, and mitochondrial dysfunction, oxygenation stress, and cellular apoptosis are important causes of PD [10-11]. Mitochondria are important organelles for intracellular energy production and play a key role in the regulation of energy metabolism and apoptosis. Their energy production relies on the transfer of electrons through the mitochondrial respiratory chain, which consists of five enzyme complexes (Complexes Ⅰ to Ⅴ). Not only does the mitochondrial respiratory chain produce energy, but it is also the main site of free radical production in the body, and damage to any of its parts will result in an increase in the production of free radicals. Complex I (NADH-CoQ reductase) is the largest and the most vulnerable one.
CoQ10 is an important component of complex I, an electron acceptor for I/II, and an important natural antioxidant that scavenges free radicals and promotes ATP production in mitochondria. Rot, a common pesticide, is a selective repressor of mitochondrial complex I and inhibits ATP production by the mitochondrial respiratory chain, which leads to oxidative stress. Oxidative stress is an important factor in the selective damage of nigrostriatal dopaminergic neurons, which can initiate PD induced by environmental factors, and can be continuously enhanced by other cellular factors. In the present study, it was observed that the intracellular ROS content of PC12 cells increased significantly after 24 h of Rot treatment, and the intracellular ROS level decreased after the administration of CoQ10, suggesting that CoQ10 can reduce the Rot-induced increase in ROS level, and that CoQ10 can play a neuroprotective role by scavenging the excessive oxygen radicals in the cytoplasm.
In the present study, we found that Rot has obvious cytotoxicity to PC12 cells, with the increase of Rot concentration, the cell damage becomes more obvious, the synapses are shortened or even disappeared, and the cell survival rate is reduced, and CoQ10 can improve the damage and apoptosis of PC12 cells caused by Rot. PD is a series of pathological processes caused by pathological apoptosis, which can be induced by both endogenous and exogenous pathways. At least three pathways have been identified to be involved in cell death, namely, the mitochondrial pathway, the death receptor pathway, and the endoplasmic reticulum pathway, of which the mitochondrial pathway is the most classical[12-13] . The Bcl-2 protein family located in the mitochondrial membrane plays a crucial role in the regulation of cellular apoptosis. The Bcl-2 protein family is divided into anti-apoptotic proteins and pro-apoptotic proteins, with the former including Bcl-2, Bcl-xl, etc., and the latter including Bax, Bad, etc. The mitochondrial pathway is the most classic one [12-13].
It has been shown that Bcl-2 can prevent neuronal apoptosis by resisting the toxicity of intracellular ROS[14] . Elevated Bcl-2/Bax expression ratio can protect cells from apoptosis.Caspas e-3 is the execution protein of apoptosis, including the inactive precursor procaspas e-3 and the activated active Caspas e-3, which is found in very low levels in normal cells. Under physiological conditions, AIF exists between the inner and outer mitochondrial membranes and is a mitochondrial oxygen reductase. When stimulated by apoptosis, it will be transferred to the nucleus through the nuclear localization sequence, causing chromosome condensation and DNA damage, and initiating apoptosis that does not depend on caspases[15-16] . In this experiment, we found that the expression ratio of Bcl-2/Bax in PC12 cells after Rot treatment was down-regulated, and active caspase-3, which was originally low, was also expressed in large quantities.CoQ10 could increase the expression ratio of Bcl-2/Bax, i.e., it could up-regulate Bcl-2 and down-regulate Bax, and at the same time, it could reduce the expression of active caspase-3.
In summary, the protective effect of soluble CoQ10 on Rot-PC12 cell apoptosis may be achieved by scavenging intracellular oxygen radicals to improve the oxidative stress status, up-regulating Bcl-2 and down-regulating Bax, and decreasing the expression of active Caspas e-3 and Caspas e-9.
References .
[1] SUBEDI L, KWON OW, PAK C, et al . N,N-disubstituted az - ines attenuate LPS-mediated neuroinflammation in micro glia and neuronal apoptosis via inhibiting MAPK signaling pathways [J] . . BMC Neurosci, 2017 , 18(1) :82 .
[2] ARAWAKA S, FUKUSHIMA S, SATO H, et al . Zonisamide attenuates α-synuclein neurotoxicity by an aggregation-inde - pendent mechanism in a rat model of familial Parkinson's dis - ease[J] . PLoS One, 2014, 9(2) :e89076 .
[3] BERGAMINI C, MORUZZI N, SBLENDIDO A, et al . A wa - ter soluble CoQ10 formulation improves intracellular distribu - tion and promotes mitochondrial respiration in cultured cells [J] . PLoS One, 2012;7(3) :e33712 .
[4] STORCH A, JOST WH , VIEREGGE P , et al . Randomized, double-blind, placebo-controlled trial on symptomatic effects of coenzyme Q(10) in Parkinson disease[J] . Arch Neurol, 2007 , 64(7) :938-944 .
[5] MA D, STOKES K, MAHNGAR K, et al . Inhibition of stress induced premature senescence in presenilin-1 mutated cells with water soluble Coenzyme Q10[J] . Mitochondrion , 2014 , 17 : 106-115 .
[6] sanders lh, greenamyre jt . Oxidative damage to macro - molecules in human Parkinson disease and the rotenone model[J] . Free Radic Biol Med, 2013 , 9(62) :111 -120 .
[7] VOSHAVAR C, SHAH M , XU L , et al . Assessment of pro - tective role of multifunctional dopamine a gonist D-512 against oxidative stress produced by depletion of glutathione in PC12 cells . Implication in neuro protective therapy for Parkinson 's disease[J] . Neurotox Res, 2015 , 8(4) :302-318 .
[ 8] ZHAO XE, ZHU S, YANG H, et al . Simultaneous determina - tion of amino acid and monoamine neurotransmitters in PC12 cells and rats models of Parkinson's disease using a sensitizing derivatization reagent by UHPLC-MS/MS[J] . J Chromato gr B Analyt Technol Biomed Life Sci, 2015 , 995(996) :15-23 .
[9] SIKORSKA M, BOROWY-BOROWSKI H, ZURAKOWSKI B, et al . Derivatized alpha-tocopherol as a CoQ10 carrier in a novel wa- ter-soluble formulation[J] . Biofactors, 2003 , 18(1 -4) :173-83 .
[ 10] BUCHMAN AS, WILSON RS, SHULMAN JM, et, al . Parkin - sonism in older adults and its association with adverse health outcomes and neuro pathology[J] . J Gerontol A Biol Sci Med Sci, 2016 , 71(4) :549-556 .
[11] verma ak , raj j , sharma v , et , al . Plasma prolidase activity and oxidative stress in patients with Parkinson's disease [J] . Parkinsons Dis, 2015 ,(2015) :598028 .
[12] shi wy, cao c , liu l . Interferon α induces the apoptosis of cervical cancer HeLa cells by activating both the intrinsic mitochondrial pathway and endoplasmic reticulum stress-in - duced pathway[J] . Int J Mol Sci, 2016 , 17(11) :e1832 .
[13] kim sm, park yj, shin ms, et al . Acacetin inhibits neuro - nal cell death induced by 6-hydroxydopamine in cellular Parkin - son's disease model[J] . Bioorg Med Chem Lett, 2017 , 27(23) : 5207-5212 .
[14] mei jm, niu cs . Effects of CDNF on 6-OHDA-induced ap - optosis in PC12 cells via modulation of Bcl-2/Bax and Cas pase-3 activation[J] . Neurol Sci, 2014, 35(8) :1275-1280 .
[15] YANG R, CUI HJ, WANG H, et al . N-stearoyltyrosine pro - tects against glutamate-induced oxidative toxicity by an a popto - sis-inducing factor ( AIF)-mediated Cas pase-independent cell death pathway[J] . J Pharmacol Sci, 2014, 124(2) :169-179 .
[16] LI H , CHEN G , MA W , et al . Water-soluble coenzyme q10 inhibits nuclear translocation of apoptosis inducing factor and cell death caused by mitochondrial complex I inhibition[J ] . Int J Mol Sci, 2014, 15(8) :13388-13400 .
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