Dendrobium Nobile Polysaccharides Attenuates Ferroptosis and Improves Cognitive Function in Vascular Dementia Rats

Abstract

Introduction

To investigate the neuroprotective effect and mechanism of Dendrobium nobile polysaccharide (DNP) on vascular dementia (VD) rats.

Methods

VD model rats were prepared by permanent ligation of bilateral common carotid arteries. Cognitive function was tested by morris water maze test, mitochondrial morphology and ultrastructure of hippocampal synapses were tested by transmission electron microscopy, GSH, xCT, GPx4, and PSD-95 expressions were tested by western blot and PCR.

Results

The number of platform crossing was significantly increased, and the escape latency was significantly shorter in DNP group. The expressions of GSH, xCT and GPx4 in the hippocampus were up-regulated in DNP group. Moreover, the synapses of DNP group were relatively intact and synaptic vesicles increased, the length of synaptic active zone and PSD thickness were significantly increased, and PSD-95 protein expression was significantly up-regulated compared VD group.

Conclusion

DNP may take a neuroprotective effect by inhibiting ferroptosis in VD.

Introduction

Vascular dementia (VD) refers to acquired intellectual and cognitive impairment syndrome caused by brain tissue damage resulted from a series of vascular factors, whose morbidity is second only to Alzheimer’s disease (AD). VD is recognized as the only type of dementia that can be prevented and reversed with early intervention. But so far, the pathogenesis of VD has not been fully elucidated, and there is no specific treatment.

VD is a heterogeneous disease, and its pathogenesis mainly includes destruction of central cholinergic system, oxidative stress injury, inflammatory reaction, cell pyrotopia, toxicity of excitatory amino acid, autophagy, intracellular calcium overload, etc.^1,2 Liu et al. ³ recently found that subcortical ischemic VD patients have abnormal iron deposition in extensive cortical areas, which leads to neuronal damage and is closely related to the severity of cognitive dysfunction. The cerebral ischemia reperfusion injury model also confirmed that iron deposition is a new mechanism of neuronal death caused by stroke. Furthemore, the use of ferroptosis inhibitors (Ferostatin-1 and Liproxstatin-1) significantly reduced infarct size and prevented sustained neuronal injury in animal models of cerebral infarction. The iron chelator DFO, a ferroptosis inhibitor, also significantly reduced ischemic reperfusion injury in animal models. ⁴ These studies suggest that ferroptosis induced by iron deposition may be a potential mechanism of neuron loss in VD, but the specific mechanism is not completely clear.

Ferroptosis is an iron-dependent regulatory cell death pattern induced by lipid peroxidation and reactive oxygen species (ROS), which is different from autophagy, apoptosis and necrosis in morphological characteristics, and is manifested by obvious shrinkage of mitochondria accompanied by thickening of membrane density, without apoptotic corpuscle, cell shrinkage, chromatin agglutination. ⁵ Biochemical characteristics are mainly proved as intracellular accumulation of iron and ROS, activation of mitogen-activated protein kinases (MAPKs) signal transduction system, inhibition of cystine/glutamate transporter system and increase of nicotinamide adenine dinucleotide phosphate (NADPH) oxidation. ⁶ Almost all the mechanisms of ferroptosis in the current studies are related to ROS. ROS accumulation in cells is one of the direct causes of ferroptosis. ⁷ ROS accumulation is caused by Fenton reaction, lipid peroxidation and glutathione (GSH) deletion of NADPH-dependent. ⁸

Studies also confirmed that glutathione peroxidase 4 (GPX4) and xCT were the key regulators of ferroptosis.^9,10 The advantages of Chinese traditional medicine in treatment of VD increasingly highlight, which has rich resources, multiple targets, and the small side effects.^11-13 Dendrobium nobile polysaccharides (DNP) is a perennial epiphytic herb of dendrobium of orchidaceae. Its main medicinal components are polysaccharide and dendrobium alkaloid. Li et al. reported that dendrobium alkaloid can significantly improved the neurobehavioral function by suppressed p38 MAPK and NF-κB pathways. ¹⁴ Moreover, Many studies confirmed that DNP has anti-tumor, anti-oxidative stress, anti-inflammatory, anti-lipid peroxidation and immunological regulation effects.^15-17 Liu et al. ¹⁸ demonstrated that DNP can significantly reduced cerebral ischemic injury by downregulated the expression of miR-134 and inhibited inflammation and apoptosis in vivo ischemia/reperfusion and in vitro oxygen glucose deprivation/reperfusion (OGD/R) model.

Object to investigate whether DNP has a neuroprotective efect on VD rats and its possible mechanisms. In this research, modifed bilateral common carotid artery occlusion was performed, and morris water maze was used to assess whether DNP can improve cognitive function of VD rats. Meanwhile, efects of DNP on ferroptosis and synaptic plasticity of VD rats were detected to reveal the possible molecular mechanisms, which laid a theoretical foundation for the application of DNP in the treatment of VD and provided new ideas for drug therapies.

Materials and Methods

Medicine, Animals and Treatment

Dendrobium nobile was purchased from Dendrobium nobile planting base in Chishui, Guizhou, China, and was identified as the stem of Dendrobium nobile by the Laboratory of Crude Medicine, College of Pharmacy, Guizhou University of Traditional Chinese Medicine. The polysaccharide of Dendrobium nobile was extracted by our laboratory. The polysaccharide was extracted by water extraction and alcohol precipitation method, purified by DEAE sepharose Fast Flow column, and the content of polysaccharide was determined to be 98.1% by phenol-sulfuric acid method.

Seventy-two 8-week-old male Sprague–Dawley (SD) rats weighing about 220-250g.They were kept in the right temperature, the day/night ratio of 12:12 animal housing. SD rats were randomly divided into sham, VD or DNP group. The rats were anesthetized with 1% sodium pentobarbital (40 mg/kg, intraperitoneal injection), and the VD animal model was prepared by permanent ligation of bilateral common carotid arteries. ¹⁹ In the sham group, rats only performed with separated common carotid artery and vagus, without clip. In DNP group, 100 mg/kg/d DNP (powder, purity 98%) was dissolved in saline solution and administered by intragastric ingestion (i.g), which was bought from Alfa company (ChengDu, China). Ultimately, we feed rats for 3 months and then killed rats by cervical dislocation, took samples.

Morris Water Maze

The Morris Water Maze (MWM) is used to detect rodent^,s cognitive function, which is composed of two tests: a positioning sea trial and a space exploration trial. During the positioning sea test, rats were placed into the water facing the pool wall from four entrance, and then entered the water from four quadrants in the four tests. The time stayed on the platform of rats for 10 seconds was defined as successful platform search. The device of MWM can automatically record the time from entering the water to find the platform successfully, which also means escape incubation period. The space exploration trial is utilized to measure the memory of rats for the original platform. In the space exploration experiment, the platform was removed after finished positioning navigation experiment, and then rats were put into the pool at any entrance, and their swimming track in a certain period of time was recorded.

Real-time PCR (RT-PCR)

After behavioral test, 6 rats in each group were decapitated, and their hippocampal tissues were separated, and then frozen with liquid nitrogen and stored at −80°C. For each sample, 2 μL cDNA was quantified and duplicated using SYBR Select Master Mix for CFX on a Light Cycler 480 II according to per manufacturer’s instructions. Circulation conditions: 2 min at 50°C, 2 min at 95°C, 40 cycles at 95°C for 15 s, 1 min at 60°C. The melting curve is cycled immediately under the following conditions: 30 s at 95°C and 15 s at 60°C. Melting curve analysis was performed to verify primer specificity. Data are showed as a fold change above the mRNA levels of proliferative condition using 2^−∆∆Ct values. Primer design and synthesis of all target genes xCT, GPx4, and β-actin were obtained from the database (https://pubmed.ncbi. NLM. Nih.gov/). Primer design and synthesis were carried out by Shenggong (Shanghai, China). Primer sequence:

xCT

Forwardprimer 5′-ATC AAC CGA GGG TGC CAA CA -3′

Reverseprimer 5′-GAG GTT CTT CCC CAG CTT GT -3′

GPx4

Forwardprimer 5′-CCG TCT GAG CCG CTT ATT -3′

Reverseprimer 5′-CAC GCA ACC CCT GTA CTT AT -3′

β-actin

Forwardprimer 5′-GCC ATG TAC GTA GCC ATC CA -3′

Reverseprimer 5′-GAA CCG CTC ATT GCC GAT AG -3′

Western Blotting

Hippocampal tissue removed from storage at − 80°C was dissolved with RIPA lysis buffer containing a mixture of protease inhibitors and phosphatase inhibitors. The solution was centrifuged at 12000 g for 10 min and then supernatant were analyzed. Protein concentration was determined using the BCA Protein analysis kit (Thermo Fisher Scientific, Inc.). A total of 25 μg of proteins were isolated using 10% or 12% SDS polyacrylamide gel and transferred to PVDF membrane. After sealing with 5% skim milk for 1 h, the membrane was incubated with primary antibodies overnight on a shaker screen. Primary antibodies contained rabbit anti-xCT (1:500 dilution, Boster, BM5318), rabbit anti-Gpx4 (1:1000 dilution, Proteintech, Cat No: 67763-1-Ig) and rabbit anti-PSD-95 (1:2000 dilution, Abcam, ab238135). Rabbit-anti-GAPDH (1:1000, Beyotime, AF0006) for load control. After washing in Trisbufered brine, the membranes were incubated with corresponding secondary antibodies (diluted at 1:5000, Abcam, Cambridge, UK). Cross reactivity was observed using western blotting assay of ECL (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and then Lab imaging system was analyzed by scanning optical density analysis.

Transmission Electron Microscopy (TEM)

After the behavioral test, 6 rats in each group were rapidly cut into 1 mm×1 mm×1 mm pieces from hippocampus tissue and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB) (pH 7.4) for 2 h at room temperature. It was then fixed in 1% osmium tetroxide in .1 M PB, following with dehydrated in graded ethanol and embedded in a beam capsule. Slices of 50-70 nm thick were cut from the embedded tissues and collected on the grid to dry overnight. The grids were stained with 2% uranyl acetate for 30 min and lead citrate for 15 min, and then observed under a transmission electron microscope (JEOL, JEM 1400 PLUS).

Statistical Analysis

Statistical analysis was performed using SPSS Version 21.0 software for Windows (SPSS Inc., Chicago, IL, USA). Results were expressed as mean ± standard deviation (SD). The differences among groups were analyzed by one-way analysis of variance (ANOVA). Then the Student-Newman-Keuls (SNK) test was used was used for comparison between groups. Significance set to P < .05.

2.7 Ethical approval. All animal procedures were accordance with Declaration of Helsinki, and were contingent on the Ethics Committee of Gannan Medical University and were executed in line with the rules of the Care and Use of Laboratory Animals (granted number was GYYFY-2021-011). This article does not incorporate any studies with human participants.

Results

Effects of DNP on Behavioral Ability of VD Rats

The escape latency time within 5 days of the training test was recorded. The escape latency of rats in VD group was significantly longer than that in SHAM group (P < .01), indicating that the cognitive function of rats in VD group was impaired. After DNP treatment, escape latency decreased significantly (P < .05) (Figure 1A). In space exploration experiment, the number of platform crossing in VD group was significantly reduced compared with SHAM group (P < .01), indicating that the memory function of VD group was significantly impaired. Compared with VD group, the number of platform crossing in DNP + VD group was significantly increased (Figure 1B, P < .05), but still less than that in Sham group.

Figure 1.

Effects of DNP on behavioral ability of VD rats. (A, D): The escape latency of rats in each group. (B): The number of times the rats crossed the platform in each group. (C): Swimming track of searching platform in each group. (**P < .01, *P < .05 vs VaD group, n = 6 rats in each group).

In addition, at the beginning of the positioning navigation experiment, the way of finding the platform of each group was random, and the swimming route was concentrated in the edge region. With the extension of training time, the motion trajectory of Sham and DNP + VD group was purposeful and trend, while the rats in VD group were still random and marginal until the end of training (Figure 1C).

Effects of DNP on Mitochondrial Morphology of Hippocampal CA1 Area in VaD Rats

TEM was used to observe the morphological changes of mitochondria in hippocampal CA1 tissue (Figure 2). As can be seen from the figures, the morphology of mitochondria in the SHAM group was normal, and the mitochondrial membrane, mitochondrial crista were intact (Figure 2A). The overall morphology of mitochondria in VD group was changed, mitochondrial membrane was destroyed and mitochondrial crest almost disappeared (Figure 2B). The mitochondrial membrane and mitochondrial crista in DNP + VD group were still incomplete, but improved compared with VD group (Figure 2(C)). The results showed that DNP intervention could improve mitochondrial morphological destruction in VD hippocampal tissue.

Figure 2.

Effects of DNP on mitochondrial morphology in hippocampal CA1 area of VD rats. (A): Mitochondrial membrane, mitochondrial cristae were intact in SHAM group. (B): Mitochondrial membrane was destroyed, mitochondrial cristae almost disappeared and number of mitochondrial cristae was decreased in VD model group. (C): Mitochondrial membrane rupture and cristae reduction were rescued by DNP. (×50,000) (Scale bar = 200 nm)(n = 40 mitochondria).

Effects of DNP on GSH, xCT and GPx4 Expression in VD Rats

We detected the expression of ferroptosis markers GSH (Figure 3A), GPx4 (Figure 3B and 4C) and xCT (Figure 3C and 4B) in the hippocampus after rats modeling. Compared with SHAM group, the expression levels of GSH, xCT and GPx4 in VD group were significantly decreased (P < .01). After DNP treatment, the expressions of GSH, xCT and GPx4 were up-regulated (P < .01), suggesting that DNP could inhibit ferroptosis by up-regulating the expressions of GSH, xCT and GPx4 in VD rats.

Figure 3.

Effects of DNP on expression of ferroptosis markers in hippocampus of VD Rats. (A): GSH content in each group. (B): GPx4 mRNA expression in each group. (C): xCT mRNA expression in each group. (**P < .01, *P < .05 vs VD group, n = 6 rats in each group).

Figure 4.

Effects of DNP on xCT and GPx4 protein expression in hippocampus of VD rats. (A): Western blot detected the expression of xCT and GPx4 in hippocampus of VD rats. (B-C): The quantitative detection of xCT and GPx4 protein. (**P < .01, *P < .05 vs VD group, n = 6 rats in each group).

Effects of DNP on Synaptic Ultrastructure and PSD-95 protein Expression in Hippocampus of VaD Rats

To investigate the effects of DNP on synaptic damage in VD rats, the ultrastructure of hippocampal synapses in each group was observed by TEM, and the expression level of PSD-95 was detected by western blot. The results showed that compared with SHAM group, the presynaptic membrane was swollen, synaptic space was blurred, synaptic vesicles were sparse, PSD was thinner, and the length of synaptic active zone (SAZ) was shortened in VD group. Compared with VD group, the synapses of DNP + VD group were relatively intact and synaptic vesicles increased, SAZ length and PSD thickness were significantly increased, and PSD-95 protein expression was significantly up-regulated (Figure 5A-D).

Figure 5.

Effects of DNP on synaptic ultrastructure in hippocampus of VD rats. (A): The hippocampal CA1 area of rats in each group was observed by TEM (×50,000). (Scale bar = 300 nm)(n = 40 synapses). (B): SAZ length of hippocampal synapses in each group. (C): PSD thickness of hippocampal synapses in each group. (D): The expression of PSD-95 protein was detected in hippocampus of VD rats (n = 6 rats per group). (**P < .01, *P < .05 vs VD group).

Discussion

Ferroptosis is a newly discovered form of cell death dependent on the generation of iron and reactive oxygen species (ROS), which is characterized by the accumulation of lipid peroxides to lethal levels. ²⁰ Glutathione peroxidase 4 (Gpx4) plays a key role in the ferroptosis by converting toxic lipid peroxides into non-toxic lipid alcohols. ²¹ System XC-light chain (xCT) is an antiporter involved in the transport of cysteine, which is an important raw material for glutathione (GSH) synthesis. ²² GSH is a reducing agent of Gpx4. When Gpx4 is inactivated, ROS aggregated and lipid peroxidation occured, which is leading to ferroptosis.^7,23 In this study, the characteristics of ferroptosis in VD hippocampal tissue were found including mitochondrial shrinkage, crest reduction or disappearance; and the expression of Gpx4 and xCT, the negative regulators of ferroptosis, were down-regulated in VD rats. These findings confirmed ferroptosis is involved in the development of VD.

Considering that ferroptosis is involved in the pathological process of VD, it is promising to develop a therapeutic approach targeting ferroptosis pathway. Whether specific inhibitors of ferroptosis could improve nerve repairment is the focus of ferroptosis research. Ferrostatin-1 is a first-generation ferroptosis inhibitor, which has been proved to inhibit ferroptosis effectively in vitro.^24,25 However, due to plasma and metabolic instability, its effectiveness is very weak in vivo. Traditional Chinese medicine (TCM) has the advantages of rich resources, small toxic and side effects, multiple channels and multiple targets. Meanwhile, nerve cell injury is characterized by multiple factors and complex mechanism, so the advantages of exploited neuroprotective drugs from TCM have gradually emerged. At present, many TCMs have been reported in treatment of nervous system diseases successfully. Dendrobium nobile Lindl.(DNL), a valuable Chinese herb, ranked first among the “nine immortal herbs in China” because of its excellent tonic properties in alleviating paralysis, lowering Qi, and tonifying the weakness of the internal organs. The main active medicinal components of DNL are dendrobiine and polysaccharide substances. Polysaccharide is an important substance for life activities, exerting antioxidant, ²⁶ anti-inflammatory, ²⁷ anti-lipid peroxidation, ²⁸ and anti-apoptotic effects.^29,30 In recent years, many scholars have used polysaccharide substances to treat various diseases effectively.^31,32 Moreover, traditional Chinese medicine (TCM) has the advantages of rich resources, small toxic and side effects, multiple channels and multiple targets. And DNP has been found to play a neuroprotective role in ischemia-reperfusion injury in rats. But, the neuroprotective effect of DNP in VD has not been reported. Therefore, in this study, polysaccharide of Dendrobium nobile was used as raw material to observe its neuroprotective effects on VD rats, and the possible mechanism was elucidated. Our results showed that the MWM score of rats in DNP + VD group increased significantly compared to that in VD group, although it was still lower than that in the SHAM group as a whole. These results suggest that DNP can promote the recovery of cognitive function in VD rats. In order to further explore the underlying mechanism of DNP, we used TEM to detect the mitochondrial morphology of hippocampal tissue and found that DNP intervention also can improve mitochondrial morphological destruction in VD. The expressions of GSH, xCT and GPx4 in the hippocampus tissue were up-regulated in DNP + VD treatment group. Moreover, the synapses of DNP + VD group were relatively intact and synaptic vesicles increased, the length of synaptic active zone and PSD thickness were significantly increased, and PSD-95 protein expression was significantly up-regulated compared VD group. Overall, our results indicated that DNP improved the cognitive function and attenuated ferroptosis in VD rats.

Conclusion

DNP may take a neuroprotective effect by inhibiting ferroptosis in VD.