Novel Phenylethanoid Glycosides Improve Hippocampal Synaptic Plasticity via the Cyclic Adenosine Monophosphate-CREB-Brain-Derived Neurotrophic Growth Factor Pathway in APP/PS1 Transgenic Mice

Abstract

Introduction

Alzheimer’s disease (AD) is a major public health concern worldwide, but there are still no drugs available that treat it effectively. Previous studies have shown that phenylethanoid glycosides have pharmacological effects, which include anti-AD properties, but the underlying mechanisms by which they ameliorate AD symptoms remain unknown.

Methods

In this study, we used an APP/PS1 AD mouse model to explore the function and mechanisms underlying savatiside A (SA) and torenoside B (TB) in the treatment of AD. SA or TB (100 mg·kg-1·d-1) was orally administered to 7-month-old APP/PS1 mice for 4 weeks. Cognitive and memory functions were measured using behavioral experiments (including the Morris water maze test and the Y-maze spontaneous alternation test). Molecular biology experiments (including Western blotting, immunofluorescence, and enzyme-linked immunosorbent assays) were used to detect any corresponding changes in signaling pathways.

Results

The results showed that SA or TB treatment could significantly reduce cognitive impairment in APP/PS1 mice. We also showed that chronic treatment with SA/TB could prevent spine loss, synaptophysin immunoreactivity, and neuronal loss in mice, thereby improving synaptic plasticity and moderating learning and memory deficits. SA/TB administration also promoted the expression of synaptic proteins in APP/PS1 mouse brains and upregulated phosphorylation of proteins in the cyclic adenosine monophosphate (cAMP)/CREB/brain-derived neurotrophic growth factor (BDNF) pathway that are responsible for synaptic plasticity. Additionally, chronic SA/TB treatment increased the levels of BDNF and nerve growth factor (NGF) in the brains of APP/PS1 mice. Both astrocyte and microglia volumes, as well as the generation of amyloid β, were also decreased in SA/TB-treated APP/PS1 mice compared to control APP/PS1 mice.

Conclusion

In summary, SA/TB treatment was associated with activation of the cAMP/CREB/BDNF pathway and increased BDNF and NGF expression, indicating that SA/TB improves cognitive functioning via nerve regeneration. SA/TB is a promising candidate drug for the treatment of AD.

Introduction

Alzheimer’s disease (AD) is an irreversible age-associated neurodegenerative disease that is characterized by progressive cognitive decline and memory loss. Typical pathological findings in AD include nerve fiber tangles, amyloid plaques, neuroinflammation, brain atrophy, and the loss of neurons and synapses [1]. Amyloid-β (Aβ) proteins are believed to be related to AD because they are abnormally aggregated in the tissue of patients with AD [2]. Although several AD drugs have been produced in recent years, treating AD remains a challenge [3, 4]. AD is characterized by gradual declines in cognitive ability, particularly in the areas of working, spatial episodic, and long-term memory [5]. Effective treatments for AD would help drastically improve these patients’ quality of life. The function of phosphorylated cyclic adenosine monophosphate (cAMP) signaling and its transcription of the downstream factor cAMP response element binding protein (CREB) have received increasing attention from AD researchers [6]. In some cases, the factors that are influenced by cAMP-CREB have also been identified as important mediators of learning and memory [7].

Monochasma savatieri Franch. ex Maxim (Scrophulariaceae) is a plant that is widely distributed in the southern Chinese provinces of Jiangsu, Zhejiang, Jiangxi, and Fujian [8]. It is a perennial herb with several Chinese medicinal properties, including Yanning granules. Many diseases, such as coughs and the common cold, can be treated using whole plants and roots [9]. Extracting elements from Monochasma savatieri Franch revealed that savatiside A (SA) and torenoside B (TB) were two major components of the phenylethanoid glycosides (PhGs) in the plant [10]. PhGs are water-soluble phenolic compounds that are present in many medicinal plants and have a wide range of applications in the treatment of diseases. They play important roles in agents which treat the influenza virus, inflammatory conditions, oxidative dysregulation, and bacterial infections [11, 12]. Our previous studies have shown that SA and TB compounds can prevent the development of AD by alleviating oxidative stress, which is induced by Aβ_25–35 via the regulation of calnexin and intracellular calcium [13, 14]. In our study, network pharmacology prediction found that PhGs may attenuate AD pathology through the cAMP-signaling pathway. Other recent studies have also suggested that traditional Chinese medicine can improve hippocampal synaptic plasticity in AD and have highlighted the role of the brain-derived neurotrophic growth factor (BDNF)/TrkB/p-CREB-signaling pathway in particular [15]. The CREB-signaling pathway is also important in cognitive functions such as memory and neuronal plasticity [16]. However, despite these previous findings, it is not known whether SA/TB can improve synaptic plasticity in APP/PS1 dementia mouse models via modulation of the cAMP/CREB/BDNF signaling pathway.

Here, we first examined the efficacy of SA and TB on APP/PS1 transgenic mice. We next aimed to clarify the potential molecular mechanisms underpinning their effects. We hope our findings can provide new perspectives on PhGs and aid in the development of a drug target for the treatment of AD.

Materials and Methods

Chemicals and Reagents

SA and TB (online suppl. Fig. S1; for all online suppl. material, see https://doi.org/10.1159/000531194) were provided by the department of natural medicinal chemistry in Soochow University, with a high purity (>98% at HPLC), as previously described [13]. Primary antibodies against p-CREB (#9198), CREB (#9104) were purchased from CST (USA). Primary antibodies against synaptophysin (ab32127) were purchased from Abcam (Cambridge, UK). Golgi staining solution (G1069), β-actin (GB11001) were purchased from Servicebio (Wuhan, China). Antibody GFAP was obtained from millipore (MAB3402, USA), and antibody Iba-1 was obtained from wako (#19741, Japan). The horseradish peroxidase (HRP) coupled with sheep anti-mouse or anti-rabbit secondary antibodies was purchased from Thermo Fisher (USA). All other chemicals used were commercially available standard chemicals.

Animals and Treatments

Thirty male APP/PS1 mice and ten male wild-type C57BL/6 mice with 7-month-old and weighing 20–22 g were purchased from GemPharmatech Co., Ltd (Nanjing, China), approval number: 2023-B10. All mice used in this study were treated according to the protocols approved by the Animal Experiment Ethics Committee, Suzhou Institute of Medicine, Chinese Academy of Sciences.

Then, thirty APP/PS1 mice (TG) were randomly divided into three groups, ten wild-type C57BL/6 mice were conducted as the negative control. Mice were given drugs or normal saline by intragastric gavage for 4 weeks, once a day. Mice in the control and model groups were given normal saline. Mice in the SA and TB groups were administered SA or TB at 100 mg/kg, respectively, as reported previously [13].

Behavioral Tests

Y-Maze Spontaneous Alternation Test

After the last administration, mice were tested with the Y-maze test to evaluate spatial recognition memory, as previously described [13]. The entry of the mouse’s hind paw into the single arm is defined as one arm entry. The tests were performed for 3 continuous days. After a single training trial, the time latency is measured (consolidation trial). The room in which tests were conducted remained quiet and dark. The number of correct responses (scored from 0 to 20) was recorded in the test.

Morris Water Maze Test

Mice were trained to swim as previously described [17]. The apparatus is composed of a round pool (120 cm in diameter, height 40 cm) and a platform in the center of the first quadrant, which filled with water at the temperature of 25 ± 1°C. Record the time for mice to locate and climb the fixed platform as the incubation period. The duration of a trial was 90 s. The escape latency of mice was observed respectively from four quadrant for 5 continuous days. If the mice could not reach the platform within 60 s, they were guided to the platform and stayed for 10 s. After 5 days of training, the platform was removed and the probe trial was conducted. Mice in each group were put into water from the same water entry point. The number of times the mice crossed the original platform and the time they stayed in the quadrant of the original platform were recorded. The locus of swimming was recorded by a computerized tracking system.

Analysis of Spines

After the experiment, the mice were sacrificed, the frontal cortex and hippocampal were fixed with 4% paraformaldehyde, then were stained with the FD Rapid Golgi Stain kit (USA). The images of stained spines were observed by using a ×100 oil-immersion objective. The sections with the same position and field of view were selected to take photos, and the image analysis software ImageJ was used for image processing, and the density of dendritic branches and dendritic spines of each section was calculated.

Image Analysis and Quantification

Five mice per group were analyzed. Aβ plaques are stained according to the instructions of the thioflavin S staining kit (Yuanye, China). Analysis was performed in the slides from the same area in each group of mice. Each hippocampal was given three images per slide from 3 areas (CA1, CA3, and dentate gyrus). The numbers and the area of Aβ plaques, the area of GFAP⁺ and Iba-1⁺ cells, and the area of the hippocampus and cortex in each image were quantified by ImageJ. Microglia and astrocytes in volume of GFAP⁺ or Iba-1⁺ signal area divided by the total area of the hippocampus to quantify.

HE and Thioflavin S Staining

HE staining was performed as reported previously [13]. To visualize Aβ protein deposition in the brain of AD mice, thioflavin S staining was used [18]. First, 0.3% thioflavin S was prepared in 50% alcohol (w/v) at room temperature with moderate stirring using a magnet stirrer. After routine dewaxing, mouse brain tissue sections were washed 3 times, 5 min/time. Then, the washed brain tissue sections were stained with DAPI dye for 8 min and incubated at room temperature for 8 min. Next, the tissue sections were washed with 80% alcohol for 10 s and stained with 0.3% thioflavin S for 10 s, repeated twice. The whole process was performed in the dark or wrapped the material in aluminum foil to prevent photobleaching.

Enzyme-Linked Immunosorbent Assay

The levels of neurotrophic factors such as BDNF, nerve growth factor (NGF), and NT-3 in mice cortex and hippocampus were analyzed. The brain tissues were homogenized in RIPA buffer containing a protease inhibitor and phosphatase inhibitor, and then were detected using the ELISA kit performed according to the manufacturer’s instructions (CUSABIO).

Western Blot

Protein concentrations were detected by BCA assay kit. Sample proteins were separated by electrophoresis buffer system using the corresponding concentration of SDS-PAGE, and then the target protein was transferred to a PVDF membrane. The membrane was blocked by 5% dry milk, thus incubated with different primary antibodies (p-CREB [rabbit, 1:1,000], CREB [rabbit, 1:1,000], and β-actin [mouse, 1:10,000]) at 4°C overnight. The membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies (1:5,000 dilution) for 1 h at room temperature. Then, the immunoblotting was determined by the enhanced chemiluminescence system. The grayscale of the blot were analyzed by ImageJ.

Statistical Analysis

Statistical analyses were conducted with GraphPad Prism 8.0 software (USA). Data were presented as the mean ± standard error of the mean, and the overall results were documented by applying the one-way analysis of variance, followed by Fischer protected least significant difference post hoc tests. p < 0.05 was considered as statistically significant.

Results

SA and TB Ameliorated Cognitive Impairments in APP/PS1 Mice

The overall flow chart of animal experiments is shown in Figure 1. We first evaluated whether SA/TB might reduce cognitive deficits in APP/PS1 transgenic mice using results from behavioral experiments. The spontaneous alternation rate of the APP/PS1 mice was remarkably lower than that of the WT mice (p < 0.05). The average spontaneous alternation rate of the SA and TB group (all p’s < 0.05) was significantly increased compared with the model group (Fig. 2c). The Morris water maze test was used to evaluate the learning and memory abilities of double transgenic mice. As shown in Figure 2a and b, there was a significant difference between the WT group and the model group in escape latency, as APP/PS1 mice failed to show learning trends (p < 0.01), but SA/TB-treated APP/PS1 mice showed similar learning trends to WT mice.

Fig. 1.

Flow chart of animal experiments. APP/PS1, amyloid precursor protein/presenilin 1 mice; NS, normal saline; i.g, intragastric gavage; WT, wild type; TG, APP/PS1 transgenic mice; MWM, Morris water maze.

Fig. 2.

The effect of SA/TB on the memory of the APP/PS1 mice in the behavioral tests. a Effects of SA/TB on the escape latency of mice in place navigation of the Morris water maze test. b Representative images of the path that the mice swam along to find the platform. c The effects of SA/TB on the spontaneous alteration in Y-maze alteration test. d, e The effects of SA/TB on the ratio of searching time in target quadrant and times of crossing platform in Morris water maze. Data are expressed as the mean ± SEM (n = 10). ^#p < 0.05, ^##p < 0.01 versus WT group, *p < 0.05, **p < 0.01 versus TG group.

The model mice also consistently stayed in the target quadrant for a shorter time period than the control mice (p < 0.05), indicating that the APP/PS1 mice had spatial memory impairment. Additionally, when compared with the model group, SA/TB-treated APP/PS1 mice had increased swimming times and platform crossing times (p < 0.05) and were even at similar levels to those of WT mice (Fig. 2d, e). These results indicated that SA/TB could improve the spatial memory capacity of APP/PS1 mice.

SA/TB Treatment Alleviates Deficits in Synaptic Plasticity in APP/PS1 Mice

Aβ-induced synaptic abnormalities are closely associated with cognitive dysfunction in AD, and synaptophysin plays a key role in the release of neurotransmitters. We used Golgi staining to examine whether SA/TB treatment might prevent the loss of spines in the brains of TG mice. The average hippocampal spine density in the TG mice was significantly lower than that of WT mice (Fig. 3a, b). In contrast, SA/TB-treated TG mice showed increased spine density in the hippocampus compared with control APP/PS1 mice (Fig. 3a, b). We stained the hippocampi of 4 groups of mice with SYN antibody, a marker of presynaptic protein, using immunofluorescence in order to investigate the effects of SA/TB treatment on synapse loss (Fig. 3c). The SYN immunoreactivity in the CA3 region of TG mice was lower than that in the WT mice. However, compared with control APP/PS1 mice, SA/TB-treated TG mice showed enhanced SYN immunoreactivity and even reached levels comparable to WT mice (Fig. 3c, d). Together, these results suggest that long-term SA/TB treatment can ameliorate synaptic plasticity damage in TG mice.

Fig. 3.

Chronic SA/TB treatment attenuates the loss of spines and synapses in APP/PS1 mice. a Representative images of spines in the hippocampus of each group of the mice. Scale bars: 20 μm. b The density of spines in the hippocampus of each group of the mice was analyzed. c The coronal sections of the hippocampus of each group of mice were stained for synaptophysin (SYN). Scale bar: 25 μm. d The mean fluorescent intensity (MFI) of SYN immunoreactivity was quantified. Data are expressed as the mean ± SEM (n = 3). ^##p < 0.01 versus WT group, *p < 0.05, **p < 0.01 versus TG group.

SA/TB Suppresses Activation of Microglia and Astrocytes in APP/PS1 Mice

In AD brains, activated microglia activated astrocytes, and chemokines that cause neuronal and synaptic dysfunction, cell death, and nerve damage, are all found in proximity to neuritic plaques [19, 20]. Modulating inflammation has gained interest as a treatment strategy for slowing the progression of AD. Therefore, we investigated whether long-term SA/TB treatment might inhibit glial cell activation in APP/PS1 mice. Polyclonal antibodies against GFAP and Iba-1, which are indications of active astrocytes and microglia, respectively. The CA1 region (Fig. 4a), CA3 region (Fig. 4b), dentate gyrus region (Fig. 4c) of the coronal part of the hippocampus were stained. At the age of 8 months, the volume of hippocampal astrocytes and microglia (Fig. 4d–f) in APP/PS1 mice were increased compared with WT mice, indicating that both hippocampal astrocytes and microglia were activated in APP/PS1 mice. Conversely, TG mice treated with SA/TB showed decreased volumes of both astrocytes and microglia compared with control APP/PS1 mice. These results suggest that SA/TB treatment attenuates glial activation which is induced by Aβ plaques.

Fig. 4.

Chronic SA/TB treatment results in a decreased activation of astrocytes and microglia in the brains of APP/PS1 mice. a–c Three coronal regions (CA1, CA3, and DG) of the hippocampus in each group of the mice were stained for GFAP, Iba-1, and 4′, 6-diamidino-2-phenylindole (DAPI). Scale bars: 100 μm. d–f The percentage of the area of astrocyte and microglia occupied in total area in the hippocampus was quantified. Data are expressed as the mean ± SEM (n = 3). ^##p < 0.01 versus WT group, *p < 0.05, **p < 0.01 versus TG group.

SA/TB Alleviates Histopathological Morphology of Hippocampal CA1 Regions and Inhibits Aβ Generation in APP/PS1 mice

HE staining was performed to monitor neuronal morphology and pyramidal cells in the hippocampal CA1 region of all mice. The results revealed that WT mice exhibited normal neuronal morphology with well-arranged pyramidal cells. In contrast, the number of pyramidal cells in TG mice was markedly decreased, and the cells were arranged in a dispersed, disordered manner. However, following SA or TB treatment, the number of pyramidal cells was increased, and the degree of cell edema was decreased (Fig. 5a).

Fig. 5.

SA/TB alleviates histopathological morphology of hippocampal CA1 regions and inhibits Aβ generation in APP/PS1 mice. a HE staining was performed to monitor neuronal morphology and pyramidal cells in the hippocampal CA1 region of mice. b The coronal sections of the hippocampus in SA/TB-treated or TG mice at 8 months of age were stained with thioflavin S against Aβ. Scale bars: 200 μm. c The numbers of Aβ plaques in the hippocampus were quantified and expressed as the amount of Aβ plaques per square millimeter. d The size of Aβ plaques in the hippocampus was quantified and expressed as the percentage of areas occupied by Aβ plaques. Data are expressed as the mean ± SEM (n = 3). ^##p < 0.01 versus WT group, *p < 0.05, **p < 0.01 versus TG group.

Thioflavin S assays were used to investigate the effects of SA/TB treatment on the formation of Aβ plaques in the hippocampus of APP/PS1 mice (Fig. 5b). Quantification analysis showed that the numbers (Fig. 5c) and size (Fig. 5d) of Aβ plaques in the hippocampus of APP/PS1 mice treated with SA/TB were reduced compared with TG mice. These results indicate that SA/TB has an inhibitory effect on Aβ plaque accumulation.

Effects of SA/TB on the cAMP/CREB/BDNF-Signaling Pathway and Neurotrophic Factors in APP/PS1 Mice

Compared with the WT mice group, the average expression of cAMP and p-CREB in the hippocampi of the model group was significantly decreased (p < 0.05). The levels of cAMP and p-CREB in the hippocampi of the SA/TB treatment group were significantly upregulated compared to the model group (Fig. 6a–c). However, we failed to observe any differences in the expression of CREB between the hippocampi of SA/TB-treated and TG mice. According to previous studies, the levels of cAMP and p-CREB are downregulated in AD. These results suggest that SA/TB could increase the levels of p-CREB and cAMP significantly in AD mouse brains.

Fig. 6.

Effect of SA/TB on cAMP/CREB/BDNF-signaling pathway in APP/PS1 mice model. a The effects of SA/TB on the content of cAMP in hippocampus of mice. b, c The representative results of p-CREB, CREB, and β-actin expressed in the hippocampus of mice in Western blot. Data are expressed as the mean ± SEM (n = 3). ^##p < 0.01 versus WT group, *p < 0.05, **p < 0.01 versus TG group.

The levels of neurotrophic factors (BDNF, NGF, and NT-3) in mouse cortices and hippocampi were analyzed using ELISA. Results showed that neurotrophic factor levels in the hippocampi and cortices of the model group were significantly lower than that in the WT group (p < 0.05). In contrast, SA/TB-treated APP/PS1 mice exhibited increased hippocampal and cortical NGF, BDNF, and NT-3 levels compared with control APP/PS1 mice (Table 1). Previous studies have shown that neurotrophic factors play an important role in improving AD. Overall, our results suggest that SA/TB could significantly increase the levels of neurotrophic factors.

Table 1.

Effect of SA/TB on the expression of neurotrophic factors (BDNF, NGF, NT-3) in the brain of APP/PS1 mice

Group	BDNF, ng/mL		NGF, pg/mL		NT-3, ng/mL
	hippocampus	cortex	hippocampus	cortex	hippocampus	cortex
WT	0.46±0.03	0.75±0.01	56.19±2.45	85.68±4.22	3.90±0.13	1.56±0.04
TG	0.21±0.01a	0.51±0.01a	36.39±1.85a	50.04±1.49a	2.45±0.17a	0.98±0.05a
TG + SA	0.31±0.01**	0.69±0.01**	46.15±2.08**	70.87±2.83**	3.00±0.08*	1.33±0.02**
TG + TB	0.38±0.01**	0.70±0.01**	49.19±1.12**	74.91±3.21**	3.03±0.07*	1.40±0.09**

The data were expressed as means ± SEM, n = 5.

*p < 0.05.

**p < 0.01 versus TG group.

^a p < 0.01 versus WT group.

Discussion

Because AD involves many factors, researchers are increasingly searching for multiple pharmacologic targets [21, 22]. We previously investigated the effects of PhGs (including acteoside, TB, and SA) on Aβ_25–35 and H₂O₂-induced cognitive dysfunction based on a previous study in ICR mice and found that PhGs effectively ameliorated Aβ_25–35 and H₂O₂-induced cognitive dysfunction in mice [13, 14]. Although some extracts have had positive effects on cognitive impairment in animal models, the exact compounds which account for these effects, as well as their mechanisms of action, need to be further investigated [13, 23, 24]. Thus, we conduct a test of behavior in a model of AD using APP/PS1 mice in order to explore the potential mechanisms of PhGs such as SA and TB in CNS disease. Our behavioral testing findings showed that SA/TB played an important role in improving learning, memory, and cognitive functioning in APP/PS1 model mice (Fig. 2). Current evidence suggests that basic neuronal signal transmission involves synapse participation. Previous groups have also demonstrated that different physical and pathological conditions can influence the synaptic structure, activity, and plasticity [25, 26]. Here, we showed that long-term SA/TB treatment improved synaptic dysfunction, learning capacity, and memory abilities in APP/PS1 mice.

Aβ deposits also cause neuronal damage. Because activated astrocytes and microglia are inflammatory mediators, they also produce other pro-inflammatory factors and neurotoxic components [27]. GFAP and Iba-1 are markers of astrocyte and microglial activation [28]. We found lower levels of Aβ plaques in the cortices and hippocampi of APP/PS1 mice compared to TG mice, perhaps because of impaired synaptic transmission. We also found that SA/TB-treated TG mice had smaller microglial and astrocytic volumes than the APP/PS1 mice. Taken together, these findings suggest that SA/TB may suppress hyperexcitability in cortical networks by reducing the density of Aβ.

Based on previous behavioral research findings, we took SA/TB-induced changes in the cAMP/CREB/BDNF-signaling pathway and the neurotrophic factor pathway as a beginning point to detect key corresponding factors. We then studied the potential mechanisms which could underlie SA/TB-mediated improvement in cognitive function in APP/PS1 mice. The secondary messenger cAMP plays a critical role in regulating neuronal morphology and physiology and thus in improving cognitive functions in the brain. Additionally, recent research has shown that astrocyte-neuron lactate shuttles mediate the cAMP-induced synaptic plasticity, which modulates memory [29]. Prior evidence has shown that Aβ downregulates the expression of BDNF by lowering CREB expression levels [30]. Studies suggest that increasing cAMP and 3′, 5′-cyclic guanosine monophosphate with phosphodiesterase inhibitors can reduce the progression of AD [31]. Neuron-synapse loss is a typical pathological feature of AD, and neuronal synapse loss or damage can affect synaptic plasticity. Thus, effectively inhibiting neuron-synaptic loss can help improve synaptic plasticity [32, 33]. SYN is a presynaptic membrane vesicle protein. The release of neurotransmitters is regulated via phosphorylation. SYN is often used as a specific marker of presynaptic terminals, and its density, distribution, and numbers can be used to measure synaptic plasticity [34]. Therefore, the upregulation of cAMP, the reversal of decreases in synaptic plasticity, and the improved cognition in APP/PS1 mice which were treated with SA/TB may be related to BDNF and NGF. SA/TB-treated TG mice also showed enhanced SYN immunoreactivity. Taken together, these results suggest that long-term treatment with SA/TB can prevent decreases in synaptic plasticity in TG mice. However, the role of the cAMP/CREB/BDNF-signaling pathway, a key upstream pathway involved in synaptic plasticity, still needs to be further explored. Meanwhile, due to the small sample size from the limited number of studies involved, the results should be further validated in high-quality studies with larger sample sizes.

Conclusion

In a summary, the present study demonstrated that long-term treatment with SA/TB improves deficits in synaptic plasticity, learning, and memory. It also reduces the density of amyloid plaques in APP/PS1 transgenic mice. Decreased amyloid beta generation and modulation of the cAMP/CREB/BDNF pathway are important mechanisms of SA/TB-mediated memory improvements in APP/PS1 mice. Overall, our study suggested that activated PhGs may be a promising compound for the treatment of AD.

Statement of Ethics

This study protocol was reviewed and approved by the Animal Experiment Ethics Committee, Suzhou Institute of Medicine, Chinese Academy of Sciences, approval number 2021-B14.

Conflict of Interest Statement

The authors have no conflicts of interest to disclose.

Funding Sources

This work was supported by the Natural Science Foundation of Jiangsu Province (SBK20200213), Suzhou introduction of clinical medical team project (SZYJTD201802), Suzhou key medical support discipline (SZFCXK202111), Suzhou High-tech District Health Talents Project SGXWS2021, Suzhou Science and Technology Plan Project (SYSD2019171, SKJY2021035, SYS2020076, SKJY2021037), and the Plan Project (NMUB2020252) from Nanjing Medical University.

Author Contributions

Shiliang Ji: data curation; formal analysis; methodology; and writing–review and editing. Yijie Wu: data curation; formal analysis; and methodology. Ruifang Zhu: methodology and writing–review and editing. Dongkai Guo: data curation; formal analysis; and methodology. Yiguo Jiang: formal analysis and methodology. Lifeng Huang: conceptualization and methodology. Xinwei Ma: writing–original draft; writing–review and editing; and funds to support. Liqiang Yu: data curation; formal analysis; and funds to support.

Funding Statement

This work was supported by the Natural Science Foundation of Jiangsu Province (SBK20200213), Suzhou introduction of clinical medical team project (SZYJTD201802), Suzhou key medical support discipline (SZFCXK202111), Suzhou High-tech District Health Talents Project SGXWS2021, Suzhou Science and Technology Plan Project (SYSD2019171, SKJY2021035, SYS2020076, SKJY2021037), and the Plan Project (NMUB2020252) from Nanjing Medical University.

Data Availability Statement

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.