Clinical efficacy analysis of coenzyme Q10 combined with flunarizine in the treatment of migraine headache

 Abstract：Objective To investigate the clinical efficacy of coenzyme Q10 combined with flunarizine in the treatment of migraine. Methods 104 migraine patients admitted to the Third People's Hospital of Mianyang City, Sichuan Province, from January to June 2023 were selected for the study, and were divided into a control group and an observation group using the randomized numerical table method, with 52 cases in each group. The control group was treated with flunarizine, while the observation group was treated with coenzyme Q10 combined with flunarizine, and both groups were treated continuously for 6 months. Compare and analyze the clinical efficacy of the two groups and the occurrence of adverse reactions during the treatment period. Cerebral hemodynamic indexes (mean blood flow velocity of the posterior cerebral artery, mean blood flow velocity of the middle cerebral artery, mean blood flow velocity of the anterior cerebral artery), oxidative stress status (oxidized low-density lipoprotein (ox-LDL), argyrophilic esterase (ArE)), migraine symptoms (duration of migraine attacks, degree of migraine pain) and the incidence of adverse reactions during the treatment period were compared in the two groups before and after 6 months of treatment. Migraine symptoms (migraine attack duration, migraine pain, migraine attack frequency), and migraine pain were assessed using a numeric rating scale (NRS).

 

Results After 6 months of treatment, the total clinical effectiveness rate of patients in the observation group was higher than that of the control group, and the difference was statistically significant (P＜0.05). There was no statistically significant difference in the total incidence of adverse reactions between the two groups (P＞0.05). After 6 months of treatment, the average blood flow rate of middle cerebral artery, average blood flow rate of anterior cerebral artery, average blood flow rate of posterior cerebral artery, ox-LDL, frequency of migraine attacks and duration of migraine attacks of the patients in the two groups were lower or shorter than those in the control group, and the differences were statistically significant (P < 0.05). After 6 months of treatment, the PON-1 and ArE of both groups increased compared with the pre-treatment period, and the observation group was higher than the control group, and the differences were statistically significant (P<0.05). The differences in NRS scores between the two groups were not statistically significant when comparing patients before treatment and after 6 months of treatment (P＞0.05). The clinical efficacy of coenzyme Q10 combined with flunarizine in the treatment of migraine is remarkable, which can effectively improve the cerebral hemodynamics and oxidative stress status of the patients, and the safety is good.

 

Migraine is a common neurological disorder that can lead to severe disability, loss of productivity, and increased economic burden on families and society [1-2]. In China, the prevalence of primary migraine is as high as 23.8% [3]. In China, the prevalence of primary migraine is as high as 23.8% [3], and migraine occurs more often in young and middle-aged people, which can seriously affect the quality of life of patients [4].

 

At present, there is no cure for migraine, and the main goal of clinical treatment of migraine is to relieve migraine symptoms. It has been reported in the literature that flunarizine is a commonly used drug in the treatment of migraine, which can inhibit the inward flow of excess calcium ions into vascular smooth muscle cells, relieve cerebral vasospasm, improve cerebral blood flow kinetics, and thus effectively relieve migraine symptoms [5-6].

 

Overseas studies have pointed out that coenzyme Q10 is an antioxidant that can be used to improve the clinical characteristics of migraine [7]. However, there is still a lack of clinical evidence to support the efficacy of coenzyme Q10 in the treatment of migraine in China. Therefore, this study investigated the clinical efficacy of coenzyme Q10 combined with flunarizine in the treatment of migraine and its effect on cerebral hemodynamics, with the aim of providing an evidence-based basis for clinical application.

 

1 Objects and Methods

1.1 Subject of the study

A total of 104 migraine patients admitted to our hospital from January to June 2023 were selected for the study, and were divided into a control group and an observation group using a randomized numeric table method, with 52 cases in each group. Inclusion criteria:

(1) The diagnostic criteria for migraine are met [8];

② Age 18-80 years old; ③ Duration of the disease ≥ 1 year, frequency of migraine attacks > 2 times / month; ④ No contraindications to the drugs used in this study. Exclusion criteria: ① headache caused by other diseases; ② combined with liquid system diseases or digestive system diseases;

(iii) Combination of other craniocerebral diseases;

④ Combined mental illness;

⑤ Combined serious heart, lung, liver, and kidney dysfunction;

(vi) Combined infectious diseases.

 

The baseline data of gender, age, disease duration, body mass index, smoking history, alcohol consumption history and comorbidities of the two groups were comparable, with no statistically significant differences (P ＞ 0.05). The study was approved by the Medical Ethics Committee of the Third People's Hospital of Mianyang City, Sichuan Province [Approval No. 2022 (18)], and all patients were informed of the study and signed an informed consent form.

 

1.2 Methodology

Patients in the control group were treated with oral flunarizine, 5 mg/times, once /d. Patients in the observation group were treated with oral coenzyme Q10 combined with flunarizine, flunarizine 5 mg/times, once /d; coenzyme Q10 10 mg/times, three times /d. Patients in the two groups were treated continuously for 6 months, and were followed up once a week during the treatment period.  All patients completed the 6-month treatment, and there was no case dropout. All patients completed the treatment for 6 months, and no case was dropped. During the acute attack of migraine, NSAIDs were given according to the "Chinese Guidelines for the Diagnosis and Treatment of Migraine (2022 Edition)"[8] .

 

1.3 Observation indicators

1.3.1 Comparison and analysis of the clinical efficacy of the two groups after 6 months of treatment  

After 6 months of treatment, cure was defined as complete disappearance of migraine and other clinical symptoms, with no recurrence one month after stopping the drug; significant effect was defined as >75% reduction in the frequency of migraine attacks and >50% shortening of migraine duration; effective was defined as 50%～75% reduction in the frequency of migraine attacks and 25%～50% reduction in migraine duration; and ineffective was defined as <50% reduction in the frequency of migraine attacks and <25% reduction in the duration of migraine [8]. Ineffective is a reduction in the frequency of migraine attacks <50% and the duration of migraine <25% [8]. Total effective rate = (number of cured cases + number of cases with apparent effect + number of effective cases) / total number of cases × 100%.

 

1.3.2 Comparison and analysis of cerebral hemodynamic indicators before and after treatment in the two groups of patients  

The mean blood flow velocity of the posterior cerebral artery, middle cerebral artery, and anterior cerebral artery were measured by Doppler color ultrasonography before and after 6 months of treatment, respectively.

 

1.3.3 Comparison and analysis of oxidative stress before and after treatment in the two groups of patients  

Before and after 6 months of treatment, 5 ml of fasting venous blood was drawn from patients of both groups, and the levels of paraoxonase 1 (PON-1) and oxidized low density lipoprotein (ox-LDL) were measured by enzyme-linked immunosorbent assay; the levels of arylesterase (ArE) were measured by colorimetric analysis. The levels of arylesterase (ArE) were determined by colorimetric analysis.

 

1.3.4 Comparison and analysis of migraine symptoms before and after treatment of the two groups of patients  

The duration of migraine attacks, the degree of migraine pain, and the frequency of migraine attacks were recorded before and after 6 months of treatment, respectively. A numerical rating scale (NRS) was used to assess the migraine pain level of the two groups, and a higher NRS score indicated more severe pain [9].

 

1.3.5 Comparison and analysis of the occurrence of adverse reactions during the treatment of the two groups of patients  

Adverse effects during the treatment period were recorded, mainly including nausea, drowsiness, fatigue, gastrointestinal discomfort, and so on.

 

1.4 Data analysis and processing

SPSS 27.0 statistical software was used to analyze the data. Measurement data with normal distribution were expressed as "x ± s", and the t-test was used to compare the means between groups; count data were expressed as cases (%), and the χ2 test was used to compare the data between groups. p < 0.05 was regarded as statistically significant difference.

 

2 Results

2.1 Comparison of the clinical efficacy of the two groups of patients after 6 months of treatment

After 6 months of treatment, the total clinical effectiveness rate of the observation group was higher than that of the control group, and the difference was statistically significant (P < 0.05). The difference was statistically significant (P < 0.05).

 

2.2 Comparison of cerebral hemodynamic indexes before and after treatment in two groups of patients

Before treatment, there was no statistically significant difference between the mean blood flow velocities of the middle cerebral artery, anterior cerebral artery, and posterior cerebral artery in the two groups (P ＞ 0.05); after 6 months of treatment, the mean blood flow velocities of the middle cerebral artery, anterior cerebral artery, and posterior cerebral artery were lower than those in the control group, with a statistically significant difference (P ＜ 0.05). The differences were statistically significant (P < 0.05).

 

2.3 Comparison of oxidative stress status between the two groups of patients before and after treatment

Before treatment, there was no statistically significant difference in PON-1, ox-LDL and ArE between the two groups (P > 0.05). After 6 months of treatment, the PON-1 and ArE of both groups increased compared with those before treatment, and the observation group was higher than the control group, with statistically significant differences (P < 0.05); the ox-LDL of both groups decreased compared with those before treatment, and the observation group was lower than the control group, with statistically significant differences (P < 0.05). The difference was statistically significant (P < 0.05).

 

2.4 Comparison of migraine symptoms before and after treatment between the two groups of patients

Before treatment, there was no statistically significant difference in the duration of migraine attacks, NRS score, and frequency of migraine attacks between the two groups (P ＞ 0.05); after 6 months of treatment, the duration of migraine attacks, NRS score, and frequency of migraine attacks of the two groups were shorter or lower than before treatment, and the duration of migraine attacks and frequency of migraine attacks of the observation group were shorter or lower than that of the control group (P ＜ 0.05). In the observation group, the duration of migraine attacks and the frequency of migraine attacks were shorter or lower than those in the control group, and the differences were statistically significant (P < 0.05). The differences were statistically significant (P < 0.05).

 

2.5 Comparison of the occurrence of adverse reactions during treatment between the two groups of patients

There was no statistically significant difference in the total incidence of adverse reactions during treatment between the two groups (P ＞ 0.05). See Table 6.

Table 6 Comparison of the occurrence of adverse reactions during treatment in the two groups [cases (%)

 

3 Discussion

Flunarizine is the first-line drug recommended by the European Headache Consortium for the treatment of migraine, and can reduce the frequency of migraine attacks by more than 50% per month [10]. Previous studies in China have shown that flunarizine can significantly reduce the frequency of migraine attacks, alleviate the pain level, and effectively shorten the duration of migraine attacks [11]. Flunarizine has a strong lipid solubility and can easily cross the blood-brain barrier to act on the cerebral blood vessels, thus inhibiting cerebral vasospasm, and can effectively improve cerebral microcirculation and neuronal metabolism, thus relieving migraine symptoms [12-13]. However, the clinical efficacy of flunarizine alone is limited, and it is often necessary to combine it with other drugs [14].

 

According to the results of this study, after 6 months of treatment, the total clinical effectiveness rate of patients in the observation group was higher than that of the control group, and the duration of migraine attacks and the frequency of migraine attacks were shorter than or lower than those of the control group, with statistically significant differences. The difference is statistically significant, indicating that the combination of coenzyme Q10 and flunarizine in the treatment of migraine is clinically effective, and can effectively shorten the duration of migraine attacks and reduce the frequency of migraine attacks. Several studies have demonstrated that coenzyme Q10 can directly participate in the reaction of peroxyl radicals, synergize with vitamin E to enhance antioxidant capacity, and enhance human immune function, improve the oxidative state of the brain, and reduce the occurrence of abnormal energy metabolism, thus suppressing migraine attacks and shortening the duration of migraine attacks [15-16]. It has been reported that coenzyme Q10 can effectively reduce the frequency and duration of migraine attacks [17]. A meta-analysis of 371 adult migraine patients showed that coenzyme Q10 could significantly reduce the duration and frequency of migraine attacks [18].

 

The results of this study showed that after 6 months of treatment, the mean blood flow velocity of middle cerebral artery, the mean blood flow velocity of anterior cerebral artery, the mean blood flow velocity of posterior cerebral artery, and ox-LDL of patients in both groups were lower than or shorter than that of the control group, and the mean blood flow velocity of PON-1, and ArE of patients in both groups were higher than those of the control group, with the difference being statistically significant.

 

Coenzyme Q10 combined with flunarizine can effectively improve cerebral hemodynamics and oxidative stress in migraine patients. Overseas studies have shown that supplementation of coenzyme Q10 can up-regulate the expression of antioxidant defense system enzymes and inhibit oxidative stress [19].

 

Studies in China have also shown that coenzyme Q10 can effectively inhibit oxidative stress in the treatment of rheumatoid arthritis and hypertension combined with arrhythmia [20-21]. The results of this study showed that there was no statistically significant difference in the total incidence of adverse reactions between the two groups. This suggests that the safety of coenzyme Q10 combined with flunarizine in the treatment of migraine is better.

 

In conclusion, the clinical efficacy of coenzyme Q10 combined with flunarizine in the treatment of migraine headache is remarkable, and it can effectively improve the cerebral hemodynamics and oxygenation stress status of the patients, and the safety is good. However, the sample size of this study is small and the follow-up period is short, so the long-term efficacy of coenzyme Q10 combined with flunarizine in the treatment of migraine has not been clarified, and needs to be further investigated in the future.

 

References:

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[8] Chinese Association of Physicians, Neurologists Branch, Headache and Sensory Disorders Committee of the Chinese Society of Research Hospitals. Chinese migraine diagnosis and treatment guidelines (2022 edition)[J]. Chinese Journal of Pain Medicine, 2022, 28(12): 881-898.

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[11] CHEN Jie, TIAN Huijuan, ZHANG Wei, et al. Effects of Chuanxiong Qingcheng Granules combined with flunarizine on cranial hemodynamics and levels of ET-1, 5-HT and CGRP in migraine patients[J]. Guizhou Medicine, 2022, 46(2): 302-303.

[12]LI W, LIU R T, LIU W D, et al. The effect of topiramate versus flunarizine on the non-headache symptoms of migraine[J]. Int J Neurosci, 2023, 133(1): 19-25.

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[14] LI Yuyu, MA Bo, XU Li. Study on the effect of heptaphylloside sodium tablets combined with flunarizine hydrochloride in the treatment of migraine[J]. Chinese Family Medicine, 2022, 20(7): 1102-1105.

[15] PAROHAN M, SARRAF P, JAVANBAKHT M H, et al. Effect of coenzyme Q10 supplementation on clinical features of migraine: a systematic review and dose- response meta-analysis of randomized controlled trials[J]. Nutr Neurosci, 2020, 23(11): 868-875.

[16]MANTLE D, HEATON R A, HARGREAVES I P. Coenzyme Q10, ageing and the nervous system: an overview[J]. Antioxidants (Basel), 2021, 11(1): 2.

[17] LUO Guochen, ZHAO Ruichao.  Effects of oral coenzyme Q10 on inflammatory factors and headache symptoms in migraine patients[J]. Medical Theory and Practice, 2021, 34(3): 410-412.

[18]SAZALI S, BADRIN S, NORHAYATI M N, et al. Coenzyme Q10 supplementation for prophylaxis in adult patients with migraine-a meta-analysis[J]. BMJ Open, 2021, 11(1): e039358.

[19]AKBARI A, MOBINI G R, AGAH S, et al. Coenzyme Q10 supplementation and oxidative stress parameters: a systematic review and meta-analysis of clinical trials[J]. trials[J]. Eur J Clin Pharmacol, 2020, 76(11): 1483-1499.

[20] Zhu Keda, Wang Rinjie, Liu Fengyun, et al. Effects of coenzyme Q10 on the levels of pro-inflammatory cytokines and oxidative stress in patients with rheumatoid arthritis[J] . Guangxi Medicine, 2020, 42(4): 417-420, 460.

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Protective Effect of Water-Soluble Coenzyme Q10 Against Apoptosis of Pc12 Cells Caused by Rotenone and Its Mechanism

 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.

 

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