Effects Of Phenylethanoid Glycosides On MDA, SOD And GSH-Px Levels in MPTP-induced PD Model Mice

WANG Xinhua (Binzhou Medical University, Yantai 264003, China)

ABSTRACT: Objective To investigate the protective effect of phenylethanoid glycosides (PhGs) on Parkinson's disease (PD) model mice induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Methods Sixty male C57BL/6 mice were randomly divided into a blank control group, model group, administration group (low dose group, middle dose group, high dose group), and positive control group (levodopa group), with 10 mice in each group. Except for the blank control group, the other groups were given MPTP intraperitoneal injections for 5 consecutive days to establish a PD mouse model. After successful modeling, the administration groups were given low, middle, and high doses of PhGs by gavage treatment. The positive control group give levodopa by gavage treatment, and the blank control group and the model group were given an equal volume of normal saline for 21 d. The behavioral test was performed at 24 h after the last administration. The mice were sacrificed 2 d after the end, and the midbrain was taken for the determination of malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and glutathione peroxidase (GSH-Px) activity. Results The behavioral test results showed that the suspension test score of the model group was significantly lower than that of the blank control group, the drop latency was shorter than that of the blank control group (P<0.001); the suspension test scores of the middle dose group and the high dose group were significantly higher than those of the model group (P<0.05); the suspension test score of the positive control group was significantly higher than that of the model group (P<0.01); the drop latency of the high dose group and the positive control group were significantly longer than those of the model group (P<0.05). The content of MDA in the model group was significantly higher than that in the blank control group (P<0.001); the content of MDA in the high-dose group was significantly lower than that in the model group (P<0.05). The activity of SOD in the model group was significantly lower than that in the blank control group (P <0.01); the activity of SOD in the high-dose group was significantly higher than that in the model group (P<0.05). The activity of GSH-Px in the model group was significantly lower than that in the blank control group (P<0.05); the activity of GSH-Px in the high-dose group was significantly higher than that in the model group (P<0.05). Conclusion PhGs can improve the motor dysfunction of PD model mice and exert neuroprotective effects. The mechanism may be related to the improvement of antioxidant capacity.

KEYWORDS: Cistanche deserticola phenylethanol glycoside; Parkinson's disease; 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine

Parkinson's disease (PD) is a neurodegenerative disease that is common in the elderly. Patients with PD usually have symptoms such as static tremors, motor retardation, muscle rigidity, and postural balance disorders [1]. The etiology of PD is multifactorial, and its pathophysiology involves various molecular and cellular mechanisms, including oxidative stress, inflammatory pathways, aquaporins, growth factors, and intestinal flora [2-5]. Currently, levodopa is commonly used in the clinical treatment of PD to improve patients' motor dysfunction by increasing the level of dopamine in the brain, but there are also certain adverse reactions [6-7]. Recent studies have shown that traditional Chinese medicine has important clinical value in the prevention and treatment of PD, with potential mechanisms including antioxidant stress, anti-neuroinflammation, and regulation of mitochondrial dysfunction [8]. Phenylethanoid glycosides (PhGs) are the main active ingredients in the medicinal materials of Cistanche deserticola, which have anti-apoptotic, antioxidant, and anti-aging effects [9]. 

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Studies have reported that total glycosides of Cistanche deserticola can reduce the accumulation of free radicals and improve the learning and cognitive function of Alzheimer's disease (AD) model mice [10]. Liu Enchong et al. [11] found that Cistanche deserticola poolside can enhance the activity of superoxide dismutase (SOD) in mouse brain tissue, and reduce the content of malondialdehyde (MDA), thereby improving cognitive dysfunction in mice. Lin Yao et al. [12] reported that Cistanche deserticola can improve behavioral disorders in PD model rats, and its protective effect on dopamine neurons may be related to inhibiting neuronal apoptosis by regulating the PI3K/Akt signaling pathway. There are few studies on the effect of PhGs on PD and its possible mechanism. Therefore, this study used C57BL/6 mice to prepare PD animal models by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to observe the effect of PhGs on PD model mice and its impact on oxidative stress-related indicators in vivo, providing experimental data for the development and application of PhGs.

1 .Material and Methods

1.1 Reagents, drugs, and main instruments

Phenylethanol glycoside is the main active component of Cistanche deserticola

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PhGs (manufacturer: Henan Hengjiuyuan Biotechnology Co., Ltd.; batch number: GL-001); MPTP (manufacturer: Sigma Corporation of the United States; batch number: M0896); Levodopa Tablets (manufacturer: Shanghai Fuda Pharmaceutical Co., Ltd.; approval number: GYZZ H31020888); MDA kit (manufacturer: Nanjing Jiancheng Technology Co., Ltd.; batch number: 20201125); SOD kit (manufacturer: Nanjing Jiancheng Technology Co., Ltd.; batch number: 20201125); Glutathione peroxidase (GSH-Px) kit (manufacturer: Nanjing Jiancheng Technology Co., Ltd.; batch number: 20201125). XR1514-RZPM rotary rod fatigue tester (manufacturer: Shanghai Xinruan Information Technology Co., Ltd.); T6 Xinyue Visible Spectrophotometer (manufacturer: Beijing Puxi General Instruments Co., Ltd.); TDZ5-WS high-speed centrifuge (manufacturer: Changsha Xiangyi Centrifuge Instrument Co., Ltd.); BT25S analytical balance (manufacturer: Sartorius, Germany).

1.2 Experimental animals

Eight-week-old male C57BL/6 mice without specific pathogen (SPF) were selected, weighing 22 to 25 g, and were purchased from Sperford (Beijing) Biotechnology Co., Ltd. [Batch No.: SCXK (Beijing) 2019-0010]. Before modeling, adaptive feeding should be conducted for 1 week, with the feeding environment at room temperature of 21~25 ℃ and standard humidity of 55%~60%. Free drinking, eating, and cage feeding should be conducted. Animal experiments were approved by the hospital ethics committee.

1.2.1 Animal grouping and modeling.

Sixty male C57BL/6 mice were randomly divided into a blank control group, model group, administration group (low-dose group, medium-dose group, high-dose group), and positive control group (levodopa group), with 10 mice in each group. Reference [13] for modeling methods: Except for the blank control group, mice in other groups were intraperitoneally injected with MPTP at a dose of 30 mg/kg for 5 consecutive days to establish subacute PD animal models.

1.2.2 Method of administration.

On the second day after the end of modeling, mice in the low-dose group, the medium-dose group, and the high-dose group were given 20 mg/(kg · d), 40 mg/(kg · d), and 80 mg/(kg · d) of PhGs by gavage, while mice in the levodopa group were given 65 mg/(kg · d) of levodopa by gavage, while mice in the blank control group and model group were given an equal volume of physiological saline by gavage for 21 consecutive days.

Cistanche deserticola experiment

1.3 Behavioral testing

1.3.1 Suspension experiment.

Using a self-made suspension test box, place the two front paws of the tested mouse on a metal rod with a diameter of 1.5 mm, a length of 30 cm, and a height of 25 cm, and record their grip. Scoring criteria: Both rear claws can grasp the metal rod, 4 points recorded; One rear claw can grasp the metal rod, 3 points recorded; If neither of the two rear claws can grasp the metal rod, score 2 points; If there is a phenomenon that the rear claw repeatedly grasps the metal rod and can grasp it intermittently, score 1 point. Each mouse was scored three times, taking the average value [14-15].

1.3.2 Rotary rod test.

Before the experiment, the mice were placed on the rotating rod of the fatigue tester and acclimated to the environment for 30 seconds. Start the fatigue tester, make the rotating speed of the rotating rod reach 26 r/min, and the infrared device records the time when the mouse moves on the rotating rod when falling from the rotating rod within 300 seconds, as the falling latency of the mouse rotating rod experiment, that is, the time when the mouse falls off the rotating rod for the first time. Each mouse was tested for three drop latency intervals of at least 30 minutes, taking the average value.

1.4 Index detection

On the second day after the completion of behavioral testing, all mice were killed by cutting off their necks, and their brains were quickly removed. The midbrain was isolated from the ice and made into 10% tissue homogenate. The supernatant was centrifuged and taken. The MDA content, SOD activity, and GSH-Px activity in the midbrain were measured in strict accordance with the instructions of the kit.

1.5 Statistical Methods

SPSS20.0 statistical software was used to analyze the data, and the measurement data conforming to the normal distribution were used with x! "± s" means that the paired comparison of multiple cost averages was conducted using one-way ANOVA, and the difference of P<0.05 was statistically significant.

2. Results

2.1 Comparison of suspension test scores of six groups of mice

The suspension test score in the model group was significantly lower than that in the blank control group (P<0.001); The suspension test scores in the mid-dose and high-dose groups were significantly higher than those in the model group (P<0.05); The suspension test score of the positive control group was significantly higher than that of the model group (P<0.01); There was no statistically significant difference in the suspension test scores between the low dose group and the model group (P>0.05). See Figure 1.

2.2 Comparison of fall latency of six groups of mice

The drop latency in the model group was significantly shorter than that in the blank control group (P<0.001); The fall latency of the high-dose group and the positive control group was significantly longer than that of the model group (P<0.05); There was no significant difference in the fall latency between the low dose group and the medium dose group compared to the model group (P>0.05). See Figure 2.

Figure 1   Comparison of suspension test scores of six groups of mice

Note: Compared with the blank control group, * * * P<0.001; Compared with the model group, # # P<0.01, △ P<0.05.

Figure 2    Comparison of fall latency of six groups of mice

Note: Compared with the blank control group, * * * P<0.001; Compared with the model group, # P<0.05.

2.3 Comparison of MDA content in the midbrain of six groups of mice

The MDA content in the model group was significantly higher than that in the blank control group (P<0.001); The MDA content in the high dose group was significantly lower than that in the model group (P<0.05); There was no statistically significant difference in the MDA content between the low dose group and the middle dose group compared with the model group (P>0.05). See Figure 3.

Figure 3    Comparison of MDA content in the midbrain of six groups of mice

Note: Compared with the blank control group, * * * P<0.001; Compared with the model group, # P<0.05.

2.4 Comparison of SOD activity in the midbrain of six groups of mice

The SOD activity in the model group was significantly lower than that in the blank control group (P<0.01); The SOD activity in the high-dose group was significantly higher than that in the model group (P<0.05); There was no significant difference in SOD activity between the low dose group and the middle dose group compared with the model group (P>0.05). See Figure 4.

Figure 4    Comparison of SOD activity in the midbrain of six groups of mice

Note: Compared with the blank control group, * * P<0.01; Compared with the model group, # P<0.05.

2.5 Comparison of GSH-Px activity in the midbrain of six groups of mice

The activity of GSH-Px in the model group was significantly lower than that in the blank control group (P<0.05); The activity of GSH-Px in the high-dose group was significantly higher than that in the model group (P<0.05); There was no significant difference in GSH-Px activity between the low dose group and the medium dose group compared with the model group (P>0.05). See Figure 5.

Figure 5    Comparison of GSH-Px activity in the midbrain of six groups of mice

Note: Compared with the blank control group, * P<0.05; Compared with the model group, # P<0.05.

3. Discussion

PD is the second most common neurodegenerative disease in the world. There is increasing evidence that oxidative stress plays a key role in the death of dopaminergic neurons, and inhibition of oxidative stress is a target for the treatment of PD. The main characteristics of oxidative stress are an increase in the content of reactive oxygen species (ROS) and a decrease or dysfunction of the antioxidant system against free radicals [16]. During the pathogenesis of PD, abnormal dopamine metabolism and mitochondrial damage in dopaminergic neurons can lead to an increase in ROS, which can damage the cell membrane and nucleic acid structure, affect the cell transcription and translation process, and cause denaturation of synthesized proteins, ultimately leading to neuroinflammation and damage to dopaminergic neurons, gradually inducing neuronal degeneration [17]. Therefore, clearing excess ROS from brain tissue is one of the methods to alleviate PD symptoms. Neurotoxins that induce irreversible PD-related effects through oxidative stress include MPTP, paraquat, rotenone, and others. Currently, MPTP, which is widely used, can be ingested through dopamine transporter proteins, inhibit the activity of mitochondrial complex I, cause neuronal degeneration and necrosis, and better simulate the pathophysiological changes and symptoms of movement disorders in PD [18].

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Cistanche deserticola ma know as"desert ginseng",is cistanche safe,is a dried, scaly, fleshy stem of the Orobanchaceae plant Cistanche deserticola, which has the effects of tonifying the kidney, strengthening yang, moistening the intestines, and relieving constipation. PhGs are the main active ingredients in the medicinal material of Cistanche deserticola, and are also the main monitoring indicators for quality control of Cistanche deserticola [19]. Previous studies have shown that PhGs have a wide range of pharmacological effects, with anti-apoptotic and antioxidant effects [20]. Tao Yicun et al. [21] reported that PhGs can reduce the water content of lung tissue in rats with high-altitude pulmonary edema, reduce the content of MDA in lung tissue homogenate, increase the activity of SOD and GSH-Px, and improve high-altitude pulmonary edema in rats. Ma Xiaoqing et al. [22] found that the total phenylethanoid glycosides of Cistanche deserticola have a certain protective effect on gentamicin-induced acute renal injury in rats, and its mechanism may be related to improving antioxidant capacity. There are few reports on the neuroprotective effects and possible mechanisms of PhGs on PD model mice.

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The behavioral test results of this experiment showed that compared with the blank control group, the suspension test scores of the model group mice were significantly reduced (P. After treatment with PhGs, the suspension test scores of mice showed an upward trend, especially at medium and high doses, with a significant effect. At the same time, the mouse fall latency also showed a dose-dependent increasing trend, with high doses being the most obvious. After treatment with levodopa, the suspension test scores of mice were significantly increased, and the fall latency was significantly prolonged. The above behavioral results suggest that MPTP can lead to decreased muscle strength and motor coordination in mice, while PhGs can improve motor dysfunction in mice and have neuroprotective effects on PD mice.

SOD and GSH-Px are important components of the body's antioxidant system, which can scavenge ROS, oxygen free radicals, and hydroxyl free radicals, and protect the structural and functional integrity of cell membranes. Free radicals act on lipids to undergo peroxidation, resulting in the final product of MDA, causing cross-linking polymerization of molecules such as proteins and nucleic acids. When lipid peroxidation in the body increases and oxygen free radicals are generated in excess, antioxidant enzymes such as SOD and GSH-Px in the body will increase, thereby eliminating excess oxygen free radicals and maintaining a dynamic balance between oxidation and oxidation resistance in the body. Studies have found that the levels of SOD activity, GSH-Px activity, and MDA in PD patients are low, and these biomarkers can be used as potential diagnostic tools for measuring oxidative stress in PD patients [23-24]. The results of this study showed that after administration of MPTP, the content of MDA in the midbrain of mice increased, while the activities of SOD and GSH-Px decreased; After 3 weeks of treatment with PhGs, the content of MDA in the midbrain of mice was significantly reduced, while the activities of SOD and GSH-Px were significantly increased. It can be seen that PhGs can enhance the level of antioxidant enzyme activity in the body of mice, eliminate the increase in free radicals caused by MPTP, reduce the damage to dopaminergic neurons, and thereby improve the motor dysfunction of MPTP model mice. The specific mechanism still needs to be further designed and explored in experiments. The loss of dopaminergic neurons in the midbrain in patients with PD leads to a decrease in dopamine levels in the striatum and motor dysfunction. Levodopa enters the body and can be converted into dopamine, supplementing the dopamine deficiency in the striatum, and playing a therapeutic role. In this study, it was found that levodopa can significantly improve motor dysfunction in PD model mice, but has a small impact on oxidative stress indicators, which may be related to its specific mechanism of action.

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In summary, PhGs can improve the motor dysfunction of MPTP-induced PD model mice and exert neuroprotective effects. The mechanism may be related to improving the body's antioxidant stress damage, and the specific signal pathway still needs further research and exploration.

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