Effects of tetrahydroxy stilbene glycoside derivatives on free radical damage and apoptosis in APP695V717I transgenic mice

ABSTRACT

Mitochondrial dysfunction leading to overproduction of oxygen free radicals is an important event in the development of Alzheimer's disease. Tetrahydroxy stilbene glycoside (TSG) is one of the main effective components of Polygonum multiflorum and has a certain free radical scavenging effect. We synthesized tetrahydroxy stilbene glycoside derivatives (Mito-TSGs) that can cross the mitochondrial membrane and may provide effective protection against Alzheimer's disease. This experiment investigates the protective mechanism of tetrahydroxy stilbene glycoside derivatives against mitochondrial free radical damage and apoptosis in APP695V717I transgenic model mice. The experimental subjects were healthy 3-month-old APP695V717I transgenic model mice, while C57BL/6J mice of the same age and genetic background served as controls. The results demonstrated that the tetrahydroxy stilbene glycoside derivatives significantly improved mouse behavioral performance. It also led to a decrease in the levels of H2O2, NO, MDA, and LD, along with an increase in LDH activity and in the antioxidant enzyme activity of SOD, CAT, and GSH-Px. Moreover, it elevated the mitochondrial membrane potential, decreased the gene and protein expression of Caspase-3 and Bax, and increased the gene and protein expression of Bcl-2. Notably, the effectiveness of tetrahydroxy stilbene glycoside derivatives was superior to that of traditional tetrahydroxy stilbene glycoside.

KEYWORDS:

• Alzheimer’s disease
• tetrahydroxy stilbene glycoside derivatives
• free radical damage
• mitochondrial targeting compounds
• oxidative stress
• apoptosis
• triphenylposphonium
• cognitive dysfunction

Introduction

Alzheimer's disease (AD) is a degenerative disease of the central nervous system in which progressive cognitive dysfunction and behavioral impairment are the main symptoms, and there is no effective treatment. The more commonly accepted mechanistic hypotheses include the Aβ hypothesis, the tau protein hypothesis, the cholinergic hypothesis, the APOE4 gene hypothesis and the oxidative stress, and mitochondrial function hypothesis [1]. In addition, inflammatory reactions and abnormal autophagy are also important events in the development of AD. In particular, studies have shown that oxidative stress and mitochondrial dysfunction can be induced by Aβ neurotoxicity, and brain tissue is attacked by oxidative stress in the early stage of AD [2,3]. At the same time, the reactive oxygen species (ROS) produced by mitochondria can trigger the accumulation of Aβ [4]. It is generally accepted that oxidative stress damage accumulates with age [5,6], and neurons are extremely sensitive to oxidative stress [7]. In addition, the brain contains relatively low levels of antioxidants and antioxidant enzymes. and therefore is not efficient in removing free radicals [8,9]. Mitochondrial dysfunction caused by oxidative stress plays an important role in the pathogenesis of Alzheimer's disease. Mitochondria are the main sites of reactive oxygen species production, and complex I and complex III of the mitochondrial electron transport chain are the main sites of their production; therefore, mitochondria are particularly vulnerable to damage by oxidative stress, while oxidative stress induces apoptosis through the formation of mitochondrial membrane pore channels and the release of proapoptotic proteins [10]. Oxidative stress leads to a decrease in mitochondrial membrane potential, mitochondrial membrane depolarization, swelling and translocation of mitochondrial substances such as cytochrome c (Cyt c) to the cytoplasm [11], to recruit cysteine aspartate protease-9 (caspase-9) and apoptosis protease activating factor-1 (Apaf-1) to form apoptotic bodies. Self-activated caspase-9, and activated caspase-9 initiates downstream caspases, such as caspase-3, to induce apoptosis.

The administration of free radical scavengers at this time can reduce nerve cell damage by reducing Αβ42-induced free radical accumulation. TSG is one of the main effective components of Polygonum multiflorum, which has a certain free radical scavenging effect [12]. It is well known that mitochondria are an important source and main target for intracellular reactive oxygen species production. Although stilbene glycosides can penetrate the cell membrane, it is difficult-for them to enter the mitochondrial membrane; therefore, we synthesized Mito-TSG based on TSG that can cross the mitochondrial membrane (the chemical formula is shown in Figure 1). The aim of this study was to investigate the mechanism of the protective effect of Mito-TSG on APP695V717I transgenic model mice. Among transgenic animal models, APP695V717I transgenic model mice are one of the closest recapitulations of Alzheimer's disease in terms of pathological characteristics, and these mice are a commonly used model of Alzheimer's disease.

Figure 1. The chemical structure of Mito-TSG.

Materials and methods

1. Ethics statement

All experimental subjects were obtained from the Better Biotechnology Co., Ltd. (License number: SCXK (Su) : 2020–0009). The protocol was approved by the local ethics committee of the Fifth Affiliated Hospital of Zunyi Medical University and was carried out in strict accordance with its guidelines. There were no restrictions on subject movement, feeding, or water intake. All subjects were anesthetized at the end of the experiment to minimize the pain. In addition, we tried to reduce the number of experimental animals used in this study.

2. Animals and groups

Sixty healthy 3-month-old APP695V717I transgenic mice were randomly divided into the model group, tetrahydroxy stilbene glycoside (TSG) group and tetrahydroxy stilbene glycoside derivative (Mito-TSG) group by the random number table method. Twenty C57BL/6J mice of the same age and background were selected as the blank control. The TSG group and the Mito-TSG group were given TSG and Mito-TSG, respectively. The drug was injected intraperitoneally at a volume of 0.4 mL/20 g once a day for 4 weeks, while the model group was given the same volume of normal saline.

3. Morris water maze experiment

The maze was a black round stainless steel pool with a diameter of 150 cm and height of 50 cm, the water depth was 30 cm, and the water temperature was maintained at (22 ± 1)°C. Four entry points were marked on the wall of the pool; thus, the pool was divided into four quadrants. The center of the first quadrant was used as the target quadrant, and a circular hidden escape platform (black platform diameter 9 cm, height 28 cm) was placed in the middle of the water. The platform was 2 cm below the water surface 2 cm. During the experiment, the wall markers of the water maze and the reference objects in the surrounding rooms remained unchanged, and were used as spatial reference clues for mice to locate the platform. A camera device was installed directly above the water maze and connected to a computer, and the movement tracks of the mice were recorded synchronously and in a timely manner through image acquisition and analysis system software (SLY-WMS2.1).

4. Transmission electron microscopy observation of mitochondrial morphology

After completing the water maze test, the mice were anesthetized with 1% pentobarbital, decapitated, fixed with 4% paraformaldehyde, rinsed, dehydrated, embedded, sectioned (60–80 nm ultrathin section), stained and observed under an electron microscope (FEI company, USA).

5. JC-1 assay for mitochondrial membrane potential

A mitochondrial membrane potential assay was performed as described in the Mitochondrial Membrane Potential Assay Kit (Nanjing Jiancheng Biological Engineering Research Institute, China) using flow cytometry. At a high mitochondrial membrane potential, JC-1 aggregated in the matrix of mitochondria, forming a polymer that could produce red fluorescence (maximum excitation wavelength 585 nm, maximum emission wavelength 590 nm). At a low mitochondrial membrane potential, JC-1, a monomer that can produce green fluorescence (maximum excitation wavelength 514 nm, maximum emission wavelength 529 nm) could not aggregate in the mitochondrial matrix. The ratio of green fluorescence intensity to red fluorescence intensity can reflect the strength of the mitochondrial membrane potential.

6. Detection of H2O2, NO and MDA in brain tissue

As quantitative indicators of ROS production, H₂O₂ and NO can be used to measure the production of H₂O₂ and NO in tissue homogenate through H₂O₂ and NO detection kits (Nanjing Jiancheng Biological Engineering Research Institute, China), as described by Peng-ChengFan [13]. The degree of lipid peroxidation in mouse blood was evaluated by MDA levels, as described by Uchiyama and Mihara [14,15], and MDA levels were measured by using a spectrophotometric diagnostic kit (Nanjing Jiancheng Biological Engineering Research Institute, China). The above operations were performed in strict accordance with the kit instructions.

7. The Lactic Acid (LD) and LDH assessment

Sample collection, preservation and determination were carried out according to the instructions of the LD and LDH determination kit (Nanjing Jiancheng Biological Engineering Research Institute, China). LD content is expressed in mmol.gprot⁻¹ and LDH activity is expressed in U·gprot⁻¹.

8. Assessment of endogenous antioxidant enzyme activity(SOD, CAT, GSH-Px)

Brain tissue was washed with prechilled PBS (phosphate-buffered saline, pH 7.4) to remove residual blood, weighed, and then cut up. The tissue and the corresponding volume of PBS (9 mL of PBS for 1 g of tissue sample) were added to the tissue grinder and ground in a full ice bath, and the homogenate was finally centrifuged at 3000 rpm for 10 min. The supernatant was collected, and the protein concentration in the supernatant was determined using a BCA analysis kit (Nanjing Jiancheng Biological Engineering Research Institute, China). SOD, CAT and GSH-Px were also tested using kits purchased from Nanjing Jiancheng Biological Engineering Research Institute, China. After the solutions were prepared according to the instructions, the reaction changes of SOD at 450 nm absorbance, CAT at 405 nm absorbance and GSH-Px at 412 nm were recorded. The results were all expressed by U·mgpro⁻¹.

9. Western blot

The brain tissue was dissolved in RIPA buffer containing PMSF and phosphatase inhibitors (Beijing Solaybao Technology Co., Ltd., China). The total protein concentration in the supernatant was determined by the BCA protein detection kit. Aliquots of homogenates with the same protein concentration were separated on 12.5% SDS-PAGE gels (Shanghai Epizyme Biomedical Technology Co., Ltd., China), transferred to PVDF membranes, soaked with TBST containing 5% skimmed milk powder (closing solution), blocked for 2 h at room temperature in a shaker, and then incubated with primary antibody at 4 °C overnight. Primary antibodies included β-actin (1:20,000), Bax(1:2,000), bcl2 (1:2,000), and Caspase3 (1:1,000). The next day, the primary antibody was washed away with TBST (5 times, 5 min/wash), the PVDF membrane was immersed in secondary antibody incubation solution (1:10,000) and incubated for 2 h in a shaker protected from light, and then the secondary antibody was washed away for observation with enhanced chemiluminescence using ECL chromogenic agent.

10. Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR)

Total RNA was extracted using TRIzol reagent (Ambion), reverse transcribed to cDNA using a two-step method, and assayed by real-time PCR using SYBR Green, The designed gene-specific primers were as follows:Bax,5′-AGACAGGGGCCTTTTTGCTAC-3′(forward),5′-AATTCGCCGGAGACACTCG-3′(reverse); Bcl-2,5′-GTCGCTACCGTCGTGACTTC-3′(forward),5′-CAGACATGCACCTACCCAGC-3′(reverse); Caspase3,5′-CTCGCTCTGGTACGGATGTG-3′(forward),5′-TCCCATAAATGACCCCTTCATCA-3′(reverse);

β-actin,5′-CACGATGGAGGGGCCGGACTCATC-3′(forward),5′-TAAAGACCTCTATGCCAACACAGT-3′(reverse).

11. Statistical analysis

SPSS 26.0 was used for statistical analyses and all the results were expressed as the means ± standard deviations (mean ± SD). Significance comparisons between groups were analyzed using one-way ANOVA, and the LSD-t test was used for two-way comparisons between groups when the variance was the same, and Tamhane's T2 was used when the variance was not the same. Values of P < 0.05 were considered statistically significant.

Results

1. Effect of Mito-TSG on behavior of mice

The experimental results showed (Figure 2) that the learning memory, spatial orientation, and working memory abilities of the model group mice were significantly reduced compared with those of the blank control group. Compared to the model group, both the TSG and Mito-TSG groups showed improved behavioral performance (P < 0.05) with shorter avoidance latencies, shorter swimming distances, and an increased number of platform crossings. Compared with the TSG group, the improvement of the mice in the Mito-TSG group was significantly better than that of the mice in the TSG group (P < 0.05).

Figure 2. Morris water maze navigation experiment was performed in blank control group, model group, TSG group and Mito-TSG group (mean ± SD, n = 20). (A) Escape latency, (B) Total distance of activities, (C) Number of platform. ^# P<0.05, comparison with control group;*P<0.05, comparison with model group;^▴P<0.05, comparison with TSG group.

2. Effects of Mito-TSG on mitochondrial morphology

According to the electron microscopy results, the blank control group had oval-shaped nuclei and dense nucleoli (Figure 3A2), and mitochondria were distributed around the nuclei with clear mitochondrial cristae and bilayer membrane structures (Figure 3A1). In the model group, the number of mitochondria around the nucleus was reduced, and the mitochondria were swollen and irregular in shape, mostly showing vacuolation (Figure 3B). Compared with the model group, the ultrastructure of the TSG group was improved, but some mitochondria were still swollen (Figure 3C), while the ultrastructural damage of the Mito-TSG group was significantly improved, with a wide distribution of organelles around the nucleus and a good condition of organelles such as the mitochondria and endoplasmic reticulum (Figure 3D).

Figure 3. Typical electron micrographs. (A) control group, (B) model group, (C)TSG group, (D) Mito-TSG group. A2, D2 × 5.0k. A1, B1, B2, C2 × 8.0k. C1, D1 × 12.0k.

3. Effect of Mito-TSG on mitochondrial membrane potential

The mitochondrial membrane potential was detected using the JC-1 method and flow cytometry. The experimental results showed (Figure 4) that the level of mitochondrial membrane potential was significantly lower in the model group than in the blank control group (P < 0.05), and the level of mitochondrial membrane potential was significantly higher in the TSG and Mito-TSG groups after drug administration treatment than in the model group (P < 0.05). The improvement of mice in the Mito-TSG group was significantly better than that of the mice in the TSG group (P < 0.05).

Figure 4. The changes of mitochondrial membrane potential in control group, model group, TSG group and Mito-TSG (mean ± SD, n = 10). (A) control group, (B) model group, (C) TSG group, (D) Mito-TSG group, (E) Changes in mitochondrial membrane potential in various groups. ^# P<0.05, comparison with control group; *P<0.05, comparison with model group;^▴P<0.05, comparison with TSG group;

4. Effect of Mito-TSG on H2O2, NO and MDA contents

The assay results showed (Figure 5) that the H₂O₂, NO and MDA contents of brain tissue were significantly higher in the model group than in the blank control group (P < 0.05). The contents of H₂O₂, NO and MDA in mouse brain tissues were reduced after administration of treatment in the TSG and TSG derivative groups compared with the model group (P < 0.05). The Mito-TSG group improved significantly better than the TSG group. (P < 0.05).

Figure 5. The changes of H₂O₂, NO and MDA in control group, model group, TSG group and Mito-TSG (mean ± SD, n = 10). (A) changes in H₂0₂, (B) changes in NO, (C) changes in MDA. ^# P<0.05, comparison with control group; *P<0.05, comparison with model group;^▴P<0.05, comparison with TSG group.

5. Effect of Mito-TSG on LD content and LDH activity

The assay results showed (Figure 6) that compared with the blank control group, the brain tissue LD content of mice in the model group was significantly higher (P < 0.05) and LDH activity was significantly lower (P < 0.05). Compared with the model group, the LD content of mouse brain tissue was significantly lower (P < 0.05) and LDH activity was significantly higher (P < 0.05) in the TSG and Mito-TSG groups after administration of the treatment. The Mito-TSG group improved significantly better than the TSG group (P < 0.05).

Figure 6. LD content and LDH activity in the control group, model group, TSG group and Mito-TSG (mean ± SD, n = 10). (A) LD content, (B) LDH activity. ^#P<0.05, comparison with control group; *P<0.05, comparison with model group;^▴P<0.05, comparison with TSG group.

6. Effect of Mito-TSG on endogenous antioxidant enzyme activities (SOD, CAT, GSH-Px)

The results of the enzyme activity assay (Figure 7) showed that the SOD, CAT and GSH-Px activities in the brain tissue of mice in the model group were significantly lower than those in the blank control group (P < 0.05). Compared with the model group, the SOD, CAT and GSH-Px activities of mouse brain tissues were significantly higher in the TSG and Mito-TSG groups after administration of the treatment (P < 0.05). The Mito-TSG group improved significantly better than the TSG group (P < 0.05).

Figure 7. The activity of SOD, CAT and GSH-Px in the control group, model group, TSG group and Mito-TSG (mean ± SD, n = 10). (A) activity of SOD, (B) activity of CAT, (C) activity of GSH-Px. ^#P<0.05, comparison with control group; *P<0.05, comparison with model group;^▴P<0.05, comparison with TSG group.

7. Effect of Mito-TSG on the protein expression of Bax, Bcl-2 and Caspase3

According to the Western blot results (Figure 8), compared with that in the blank control group, the expression of Caspase-3/β-actin and Bax/β-actin in the model group was significantly increased, while the expression of Bcl-2/β-actin was decreased (P < 0.05). Compared with the model group, Caspase-3/β-actin and Bax/β-actin expression in brain tissue was significantly lower (P < 0.05) and Bcl-2/β-actin expression was increased (P < 0.05) after treatment with TSG and Mito-TSG group administration. The Mito-TSG group improved significantly better than the TSG group (P < 0.05).

Figure 8. The protein expression of Bax, Bcl2 and Caspase-3 in control group, model group, TSG group and Mito-TSG group (mean ± SD, n = 10). (A) The results from the Western Blot, (B) Quantitative analysis for the protein level of caspase-3, (C) Quantitative analysis for the protein level of bcl-2, (D) Quantitative analysis for the protein level of bax. ^#P<0.05, comparison with control group; *P<0.05, comparison with model group; ^▴P<0.05, comparison with TSG group.

8. Effect of Mito-TSG on the gene expression of Bax, Bcl-2 and Caspase-3

According to the results of RT-qPCR (Figure 9), compared with the blank control group, the gene expression of Caspase-3 and Bax in the model group was significantly increased, while the gene expression of Bcl-2 was decreased (P < 0.05). Compared with the model group, after treatment with TSG and Mito-TSG, the gene expression of Caspase-3 and Bax in brain tissue was significantly decreased, while the gene expression of Bcl-2 was increased (P < 0.05). The improvement degree of the Mito-TSG group was significantly better than that of the TSG group (P < 0.05).

Figure 9. The gene expression of Bax, Bcl2 and Caspase-3 in control group, model group, TSG group and Mito-TSG group (mean ± SD, n = 10). (A) gene expression of bax, (B) gene expression of bcl-2, (C) gene expression of caspase-3. ^#P<0.05, comparison with control group; *P<0.05, comparison with model group; ^▴P<0.05, comparison with TSG group.

Discussion

Studies have shown that AD is caused by oxidative stress early in its pathogenesis, and there are many mechanisms of oxidative stress, including Aβ neurotoxicity, formation of neurogenic tangles, and mitochondrial dysfunction [16,17]. Mitochondria are an important source of intracellular ROS production, and the production of reactive oxygen species, including superoxide and peroxide, is inevitable [18]. Its H₂O₂ production is related to the disproportionation of O₂, which is formed by different mechanisms [19,20]. Under normal conditions, H₂O₂ is involved in the regulation of redox-sensitive signals, such as insulin/IGF1 signaling, JNK signaling and AMPK signaling, and mitochondria also receive and respond to cytoplasmic signals, through which they regulate their metabolic and redox functions [21]. H₂O₂ is also involved in the redox regulation of cytoplasmic signaling and nuclear transcriptional pathways [22]. H₂O₂ plays an important role in cell signaling, but mitochondrial dysfunction causes changes in the cellular redox environment, resulting in an increased concentration of H₂O₂ [23]. Studies have shown that excess H₂O₂ activates stress-sensitive kinases (such as JNK and IKK), causing insulin resistance and resulting in inadequate energy supply in brain tissue [24]. In addition, excess H₂O₂ mediates the activation of neuroinflammation at the molecular and cellular levels [25]. The random action of reactive oxygen species also causes lipid peroxidation, and excessive lipid peroxidation is a hallmark of most neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and many other neurological disorders. The presence of antioxidant enzyme systems such as SOD, GSH-px, and CAT, which reduce the excessive production of reactive oxygen species in the mitochondrial matrix, is essential to protect cells from lipid peroxidation [26]. Under physiological conditions, mitochondria exhibit a wide range of physiological characteristics and are regulated by nitric oxide (NO) [27]. NO can regulate the production of ATP and mitochondrial membrane potential by reversibly inhibiting the consumption of cytochrome oxidase and oxygen and play the regulatory role of NO on mitochondria. NO reacts with superoxide anions to form the strong oxidant peroxynitrite [28], which has an irreversible effect on mitochondria. NO can also exert proapoptotic or inhibitory effects indirectly through the regulation of mitochondrial function [29], and in the presence of oxidative stress in the body, excess NO induces mitochondrial dysfunction and apoptosis [30].

Mitochondria are an important source and major target of intracellular reactive oxygen species production, and if free radical scavengers are administered during the pathogenesis of AD, they will help to reduce the extent of Αβ42-induced free radical damage and thus reduce neural cell damage. TSG is one of the main effective components of Polygonum multiflorum, which has a certain free radical scavenging effect and protects mitochondrial functions [31,32]. Recent studies have shown that TSG suppressed the production of inflammatory cytokines [33,34], it also alleviated cellular oxidative stress and inflammatory damage in response to Aβ by attenuating the levels of oxidation products [35]. Although TSG can penetrate the cell membrane, it has difficulty entering the mitochondrial membrane to play the role of anti-free radical damage.

The common delocalized lipophilic cation (DLC) triphenylphosphonium cation (TPP⁺), which contains three phenyl groups in its chemical structure, makes the whole molecule highly lipid soluble [36]. Meanwhile, the positive charge on the phosphorus atom in TPP⁺ can delocalize to the three benzene rings, forming a delocalized positive charge, which drives TPP⁺ across the phospholipid membrane. These two characteristics make it highly hydrophobic and have a good intracellular membrane binding ability, and it can enter the cytoplasm driven by a transmembrane potential of up to 200 mV in mitochondria and accumulate richly in mitochondria through electrostatic interactions [37–39]. Therefore, we designed TSG derivatives (Mito-TSG) with a triphenylphosphine cation (TPP⁺) as the mitochondrial targeting group. This compound can easily pass through the mitochondrial membrane and play a better anti-free radical role by accumulating in mitochondria to mitigate the cell damage caused by oxidative stress.

In this study, we synthesized TSG derivatives (Mito-TSG). TSG is one of the main effective components of Polygonum multiflorum, which has a certain free radical scavenging effect, but it does not easily pass through the mitochondrial membrane. The derivative based on TSG is a compound that easily passes through the mitochondrial membrane, which can better play the role of anti-free radicals and reduce the tissue damage caused by oxidative stress. The results showed that Mito-TSG could significantly decrease the contents of H₂O₂, NO, MDA and LD in the brain tissue of model mice and increase the activities of SOD, CAT, GSH-px and LDH. At the same time, it could also increase the protein expression of apoptosis-related Bcl-2 in the mitochondrial pathway, reduce the protein expression of Caspase-3 and Bax, and reduce apoptosis in the mitochondrial pathway. The results showed that Mito-TSG could effectively protect the morphology and function of mitochondria in the brain tissue of model mice, eliminate excessive free radicals, reduce lipid peroxidation, enhance the activity of endogenous antioxidant enzymes, improve the antioxidant ability, and have a good brain protective effect on APP695V717I transgenic mice. Compared with traditional TSG, Mito-TSG has obvious anti-free radical activity and can be further used to prepare anti-free radical damage drugs for the treatment of Alzheimer's disease. Mito-TSG can also be used to prevent or treat the pathological state of free radical injury, such as heart, brain and respiratory system injury, caused by disease.

Conclusion

Mito-TSG can relieve oxidative stress, increase the activities of endogenous antioxidant enzymes, protect the morphology and function of mitochondria, inhibit apoptosis, and improve brain injury in APP695V717I transgenic mice. The mechanism may be related to anti-free radical damage.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data that has been used is confidential.

Additional information

Funding

This work was supported by the Science and Technology Program of Guizhou Province [Qian ke He support [2020] 4Y128]. Doctoral Start-up Fundation of Zunyi Medical University (BS2021-04).