Ginsenoside RK1 inhibits microglial activation and ROS production and alleviates vascular dementia in rats

Abstract

Although ginsenoside RK1 (RK1) possesses neuroprotective properties, it is unknown how it relates to vascular dementia (VD). The purpose of this work was to show that RK1 has a neuroprotective function in VD. First, the VD rat model was established by ligating the carotid artery with two-vessel occlusion (2-VO) surgery. RK1 was given daily for 30 days. A water maze test evaluated the learning and memory functions of the rats in each group. HE staining was used to assess the pathological damage of hippocampal tissue. The microglial marker Iba-1, proinflammatory factors [tumor necrosis factor alpha (TNF-α), Interleukin (IL)-1β, and IL-6], reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and peroxisome proliferator-activated receptor γ (PPARγ) in the hippocampal tissue were detected. The results show that RK1 reduced the escape latency of VD rats, increased the time VD rats stayed in the target quadrant and the number of times they crossed the platform, and alleviated the pathological damage of hippocampal tissue. In addition, RK1 also inhibited the activation of microglia and ROS production in the hippocampus of VD rats, reduced the content of proinflammatory factors and MDA, increased the content of antioxidant enzyme SOD, and activated PPARγ expression in hippocampal tissue. Overall, RK1 alleviates cognitive dysfunction and hippocampal tissue pathological damage in VD rats and inhibits hippocampal neuroinflammation and ROS production, which may be related to the activation of PPARγ in hippocampal tissue by RK1.

Introduction

Vascular dementia (VD) is a cerebrovascular injury disease accompanied by memory loss and cognitive dysfunction, which often occurs in the elderly.⁽¹⁾ VD can be treated with medications that improve cerebral blood circulation and cerebral metabolism,⁽²⁾ cognitive function training,⁽³⁾ and active management of factors that are strongly associated with vascular risk.⁽⁴⁾ Besides, VD is linked to cerebral hypoperfusion-related ischemia, oxidative stress, and neuroinflammation.⁽⁵⁾ Therefore, the research and development of effective drugs is crucial for the treatment of VD.⁽⁶⁾

The significance of neuroinflammation in the development of VD is becoming increasingly clear. In VD, diminished blood flow and hypoxia to the brain trigger microglial activation.⁽⁷⁾ Tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and other proinflammatory substances that promote secondary brain damage are released by activated microglia.⁽⁸⁾ Learning and memory disorders result from these proinflammatory substances’ infiltration of the white matter, which damages and kills neurons.⁽⁹⁾ Reactive oxygen species (ROS) are believed to be essential for the initial and ongoing activation of microglia.⁽¹⁰⁾ Increased ROS in microglia can trigger pathways leading to inflammation and cell death.⁽¹¹⁾ Consequently, treatment strategies that target ROS in microglia may aid in lowering neuroinflammatory damage.

Peroxisome proliferator-activated receptor γ (PPARγ) is one of several transcription factors that control microglial polarization. The central nervous system has a large amount of PPARγ, which can shield neurons by reducing inflammatory reactions.⁽¹²⁾ Numerous investigations have demonstrated that PPARγ controls microglial polarization, which in turn helps to reduce inflammation. Many degenerative neurological illnesses can be effectively treated by altering the PPARγ pathway. Reducing VD in rats by controlling microglial polarization mediated by PPARγ.⁽¹³⁾

Ginsenoside RK1 is an active ingredient in ginseng and belongs to the ginsenoside class of compounds. Ginsenosides are a class of natural active substances extracted from ginseng that have multiple biological activities, such as anti-inflammatory, antioxidant, and anti-tumor. According to earlier research, RK1 protects against nerve injury. RK1 reduces the level of ROS to alleviate cognitive impairment and pathological changes caused by Alzheimer’s disease.⁽¹⁴⁾ Through the activation of PPARγ, RK1 inhibits oxidative stress, improving diabetic endothelial dysfunction.⁽¹⁵⁾

The purpose of this work is to show the effect of RK1 on microglia activation and ROS in VD and to offer a tiny point of reference for the development of therapeutic medications for VD.

Materials and Methods

Animals

Male SPF Sprague–Dawley rats, 6 weeks old (weight 160–180 ‍g) were selected. The rats were allowed to eat and drink freely and were adaptively raised for 7 days. The 2-VO approach was used to create the VD animal model. This was the precise procedure: First, rats were anesthetized continuously with 2% isoflurane gas at 0.2 ‍ml/min. The skin was disinfected with 75% ethanol and the skin was incised in the middle of the neck, then divided the subcutaneous tissue bluntly. Surgical sutures were used to ligate the carotid artery, which pulsed at the angle between the trachea and the trapezius muscle immediately below the incision. The surgical method was the same for rats in the sham group, but the common carotid artery was exposed but not ligated. The RK1 group was given drugs by gavage for 30 days after 2-VO surgery. Four groups were created out of the rats: sham group (n = 10), VD group (n = 10), VD + RK1 (10 ‍mg/kg) (n = 10), and VD + RK1 (20 ‍mg/kg) (n = 10). The experimental process is detailed in the flow chart. The National Institutes of Health’s standards for the Care Use of Laboratory Animals were followed in all animal research. The Shenzhen Second People’s Hospital Ethics Committee gave its approval to the animal study program (Approval No. 202400162).

Water maze experiment

The water maze experiment uses a circular pool filled with opaque water (milk or non-toxic dye is added to make the water turbid) and the water temperature is maintained at around 25°C. A hidden, invisible small platform is placed on the edge of the pool, with the top of the platform about 1–2 ‍cm above the water surface. The animal can only find and stand on the platform through memory. Test of orientation and navigation: acquainted the rats with the maze, after that, with their heads pointing toward the wall, the rats were placed in one of the four quadrants of the pool. The escape latency was determined by timing how long it took the rats to climb onto the platform in less than 60 ‍s. The escape delay time would be 60 ‍s if the rats were unable to locate the platform in that time. Five days in a row were spent training the rats. Test of space exploration: on the 6th day, the platform was removed and the animal was placed in the diagonal quadrant of the original platform facing the pool wall. The system recorded the number of times the animal crossed the original platform quadrant within the specified time (60 ‍s), the time it stayed in the target quadrant, and the swimming speed.

HE staining

The hippocampal tissues of rats in each group were fixed with formaldehyde solution, embedded in paraffin, and prepared into tissue wax blocks. The paraffin blocks were cut into 4 ‍mm thick sections, stained with eosin and hematoxylin according to the HE staining process, and the photographs of hippocampus tissue were examined and captured using an optical microscope.

Immunofluorescence

Paraffin sections were dewaxed and hydrated with xylene and alcohol, respectively, and subsequently, in a microwave oven, antigen retrieval was carried out using citrate antigen retrieval solution (pH ‍6.0). Nonspecific binding sites were blocked with BSA. Incubation with primary antibody Iba1 (1:100, ab178847; Abcam, Cambridge, UK) or PPARγ (1:100, #AF6284; Affinity, Jiangsu, China) was performed overnight at 4°C. Sections were rinsed and incubated with secondary antibody (Alexa Fluor 647^®, 1:500, ab150075; Abcam) for 1 ‍h at room temperature. Sections were rinsed with PBS, counterstained with 4',6-diamidino-2-phenylindole (DAPI), mounted with anti-fluorescence quenching mounting solution, and photographed under a fluorescence microscope (Nikon, Tokyo, Japan). The fluorescence image was analyzed using Image J software (National Institutes of Health, Bethesda, MD).

Dihydroethidium staining

The brain tissue was cut into 4.0 ‍μm slices using a freezing microtome and placed on frozen microscope slides. The slices were incubated in a dark room with a physiological saline solution containing 10 ‍μmol dihydroethidium for 30 ‍min. Finally, they were stained with DAPI and observed and photographed under a fluorescence microscope.

Elisa assay

The brain tissues were homogenized and centrifuged, and TNF-α, IL-6, IL-1β, MDA, and SOD in the supernatant were detected by the corresponding kits according to the manufacturer’s instructions.

Western blot

After grinding brain tissue that had been frozen in liquid nitrogen into a powder and adding RIPA lysis buffer to extract all of the protein, the mixture was centrifuged, and the supernatant was removed so that the protein concentration could be measured using a BCA kit. The proteins were separated by SDS-PAGE gel electrophoresis and subsequently transferred to PVDF membranes, and blocked with 5% skim milk powder.⁽¹⁶⁾ After overnight incubation at 4°C with either Iba-1 (1:1,000), PPARγ (1:1,000), CD86 (1:1,000), and CD206 (1:1,000) antibody, the PVDF membrane was cleaned and treated for 1 ‍h at room temperature with a secondary antibody. Enhanced chemiluminescence was utilized to create protein bands, and ImageJ software was used to assess the bands’ gray values. The internal reference for comparative analysis was β-actin.

Statistical analysis

To examine statistical data, GraphPad Prism 8 was used. To ascertain group differences, a one-way analysis of variance (ANOVA) was followed by Tukey’s post hoc test. The p value was less than 0.05, which indicated that the differences were statistically significant.

Results

RK1 alleviates learning and memory impairment in VD rats

The water maze experiment measured the movement trajectory of rats in each group (Fig. 1A). The results of positioning cruising showed that the escape latency of rats in the VD group increased, while the escape latency of rats in the RK1 (10 ‍mg/kg) and RK1 (20 ‍mg/kg) groups decreased (Fig. 1B). The results of spatial exploration showed that the number of times the VD group rats crossed the platform (Fig. 1C) and the time spent in the target quadrant (Fig. 1D) decreased, and the number of times the VD rats given RK1 (10 ‍mg/kg) and RK1 (20 ‍mg/kg) crossed the platform and the time spent in the target quadrant increased significantly. The rats’ swimming speeds did not vary between the groups (Fig. 1E).

Fig. 1.

RK1 alleviates learning and memory impairment in VD rats. (A) Swimming trajectories of rats in each group in the water maze experiment. (B) Escape latency of rats in each group in the water maze test. (C) The time spent by each group of rats in the target quadrant in the water maze experiment. (D) The number of times each group of rats crossed the platform in the water maze experiment. (E) Swimming speed of rats in each group in the water maze test. ***p<0.001 vs sham; ^{^^}p<0.01, ^{^^^}p<0.001 vs VD.

RK1 alleviates hippocampal damage in VD rats

In the sham group, the hippocampus neurons in the CA1 region morphological structure was undamaged and well-organized. The neurons in the VD group were visibly absent, disorganized, atrophied, and vanished nucleoli. The arrangement and nuclear condensation of hippocampal neurons in the RK1 treatment group were improved (Fig. 2).

Fig. 2.

RK1 alleviates hippocampal damage in VD rats. HE staining of hippocampal in the CA1 region tissue. Red arrows represent nuclear condensation, and black arrows represent disordered neuronal arrangement. See color figure in the on-line version.

RK1 attenuates microglial activation in VD rats

The expression of PPARγ in hippocampal tissue was detected by immunofluorescence (Fig. 3A) and Western blot (Fig. 3B). The findings demonstrated that rats in the VD group had higher levels of Iba-1 expression in their hippocampal regions, while RK1 (10 ‍mg/kg) and (20 ‍mg/kg) reduced the expression of Iba-1, indicating that RK1 inhibited the activation of microglia in the hippocampus. In addition, the protein expression of microglial M1 polarization marker CD86 (Fig. 3C) and M2 polarization marker CD206 (Fig. 3D) was detected by Western blot. The results showed that the expression of CD86 protein in the VD group increased significantly, and the expression of CD206 protein increased slightly, but there was no statistical significance. This may be because microglia tend to differentiate into M2 type in the early stage of the disease to promote tissue repair and inflammation resolution. The expression of CD86 protein decreased, while the expression of CD206 protein increased in the RK1 (10 ‍mg/kg) and (20 ‍mg/kg) groups. The content of pro-inflammatory factors in hippocampal tissue was detected by ELISA experiment (Fig. 3E). The results showed that the content of TNF-α, IL-1β, and IL-6 in hippocampal tissue of rats in VD group increased, while RK1 (10 ‍mg/kg) and (20 ‍mg/kg) reduced the content of the above factors, indicating that RK1 inhibited the inflammatory response of hippocampal tissue and the effect of RK1 (20 ‍mg/kg) was better.

Fig. 3.

RK1 attenuates microglial activation in VD rats. (A) Immunofluorescence detection of Iba-1 expression in hippocampus. (B) Detection of Iba-1 expression in hippocampus by Western blot. (C) Detection of CD86 expression in hippocampus by Western blot. (D) Detection of CD206 expression in hippocampus by Western blot. (E) ELISA detection of TNF-α, IL-1β, and IL-6 levels in hippocampal tissue. ***p<0.001 vs sham; ^{^^}p<0.01, ^{^^^}p<0.001 vs VD.

RK1 alleviates ROS production in VD rats

In order to prove the effect of RK1 on oxidative stress in hippocampal tissue, the levels of ROS (Fig. 4A and B), MDA and SOD (Fig. 4C) in hippocampal tissue were measured. The findings demonstrated a substantial rise in ROS and MDA levels in the hippocampus tissue of rats in the VD group and the level of SOD decreased, while RK1 treatment reversed the above changes.

Fig. 4.

RK1 alleviates ROS production in VD rats. (A, B) Detection of ROS in hippocampal tissue by DHE method. (C) ELISA detection of MDA and SOD in hippocampal tissue. ***p<0.001 vs sham; ^{^^}p<0.01, ^{^^^}p<0.001 vs VD.

RK1 activates PPARγ expression

Rats in each group had their hippocampal PPARγ expression assessed by immunofluorescence (Fig. 5A and B) and Western blot (Fig. 5C). According to the findings, the VD group’s PPARγ expression was considerably lower, while the RK1 treatment group activated the expression of PPARγ in the hippocampus of VD rat, and RK1 (20 ‍mg/kg) had a better effect in activating PPARγs.

Fig. 5.

RK1 activates PPARγ expression. (A, B) Immunofluorescence detection of PPARγ expression in hippocampal tissue. (C) Western blot detection of PPARγ expression in hippocampus. ***p<0.001 vs sham; ^{^^^}p<0.001 vs VD.

Discussion

VD is becoming more common and has a major influence on quality of life, it is thought to be crucial to evaluate cognitive enhancement techniques for this condition. For the first time, this study verified that RK1 triggered PPARγ expression, prevented microglial activation and ROS generation, reduced hippocampus damage in VD rats, and improved the rats’ capacity for learning and memory. These findings offer compelling proof of RK1’s potential as a VD treatment medication.

According to earlier research, RK1 activates SIRT3-mediated Nrf2/HO-1 signaling to produce neuroprotective benefits against MPP⁺/MPTP-induced neurotoxicity.⁽¹⁷⁾ Through its antioxidant action, inhibition of neuroinflammation, and positive modulation of the BDNF-TrkB pathway, Rk1 has antidepressant effects.⁽¹⁸⁾ Furthermore, RK1 therapy considerably reduced oxidative stress in irradiated enterocytes.⁽¹⁹⁾ The aforementioned research has verified that RK1 can lower oxidative stress and inflammation.

Damage to hippocampal neurons is the primary pathogenic alteration brought on by VD that results in cognitive impairment.⁽²⁰⁾ According to the findings of this study, RK1 could reduce the escape latency of VD rats, increase the time VD rats stayed in the target quadrant and the number of times they crossed the platform, proving that RK1 could enhance the learning and memory ability of VD rats. In addition, RK1 improved hippocampal neuronal disarrangement and nuclear consolidation in VD rats, suggesting that RK1 has an ameliorative effect on hippocampal neuronal damage.

One of the primary immune cells in the central nervous system is microglia,⁽²¹⁾ and mounting evidence indicates that microglia-mediated neuroinflammation plays a role in the etiology and development of several neurodegenerative illnesses.⁽²²⁾ Excessive microglial activation has been shown to have neurotoxic effects,⁽²³⁾ and altering microglial activation may be a useful treatment for a number of neurodegenerative diseases.⁽²⁴⁾ In this study, it was demonstrated that RK1 inhibited microglial activation in the hippocampus, specifically by reducing the expression of Iba-1 in the hippocampus of VD rats. In addition, RK1 also reduced the levels of proinflammatory factors TNF-α, IL-1β, and IL-6 stimulated by microglial activation.

ROS is a significant factor in inflammatory diseases.⁽²⁵⁾ Prolonged or chronic ROS generation is thought to be a major cause of inflammatory illnesses, and the connection between ROS and inflammation has been thoroughly studied in a number of disease models.⁽²⁶⁾ Prior research has demonstrated that VD may cause ROS to accumulate.⁽²⁷⁾ Thus, this study investigated whether RK1 could lessen neuroinflammation by reducing the generation of ROS. The results of this study confirmed that RK1 reduced the production of ROS in the hippocampus of VD rats, reduced the content of the oxidative marker MDA, and increased the content of the antioxidant enzyme SOD.

For several central nervous system disorders, PPARγ is thought to be a promising therapeutic target.⁽²⁸⁾ PPARγ activation has been shown to produce strong neuroprotective effects in a variety of animal models of VD.⁽²⁹⁾ The control of microglia-associated neuroinflammation is intimately related to the neuroprotective actions of PPARγ.⁽³⁰⁾ In this study, PPARγ in the hippocampus of VD rats was detected, and the results of this study showed that RK1 increased the expression of PPARγ in the hippocampus of VD rats.

All things considered, our data indicate that RK1 enhances cognitive performance and reduces neuroinflammation in VD rats. These therapeutic benefits are linked to the PPARγ pathway’s activation, which inhibits microglial activation and ROS. According to these results of this study, PPARγ pathway targeting may be a promising therapeutic approach for VD, and RK1 may be a novel and potential treatment for the condition.

Author Contributions

KB and XZ designed the study, completed the experiment, and supervised the data collection. XF analyzed the data and interpreted the data. LJ prepare the manuscript for publication and reviewed the draft of the manuscript. All authors have read and approved the manuscript.

Ethics Approval

Ethical approval was obtained from the Ethics Committee of the Shenzhen Second People’s Hospital (Approval No. 202400162).

The animal experiment complies with the ARRIVE guidelines and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Availability of Data and Materials

All data generated or analyzed during this study are included in this published article. The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Conflict of Interest

No potential conflicts of interest were disclosed.