Precise Aβ clearance and antioxidant therapy in Alzheimer’s disease via photoacoustic imaging-guided palladium hydride nanosheet-mediated photothermal treatment

Inflammation and oxidative stress are critical pathological hallmarks of Alzheimer’s disease (AD). Hydrogen therapy shows promise due to its selective hydroxyl radical (∙OH) scavenging capacity, yet conventional hydrogen delivery fails to effectively accumulate in the brain due to low solubility and rapid diffusion. Here, we report ultra-small palladium hydride (PdH) nanosheets as a dual-functional nanomedicine for AD treatment. The PdH nanosheets exhibit high hydrogen-releasing efficiency via autocatalysis, enabling in situ ∙OH scavenging in the brain. Concurrently, their near-infrared (NIR) photothermal effect, degrading amyloid-β (Aβ) aggregates and transiently enhancing blood-brain barrier (BBB) permeability for improved drug delivery. In AD mice, intravenous PdH treatment combined with NIR irradiation significantly reduced escape latency in the Morris water maze, paralleling reductions in brain Aβ plaques and ROS levels. This study establishes a synergistic strategy of nanoscale hydrogen delivery and photothermal therapy, addressing the dual challenges of BBB penetration and multi-pathology intervention in AD.

Clinical trial registration number

Not applicable.

Highlights

Innovative application of nanomaterials

We innovatively use ultra - small palladium hydride (PdH) nanosheets as a dual - functional nanomedicine for Alzheimer’s disease (AD) therapy. Their self - catalyzed efficient hydrogen release enables in - situ・OH scavenging in the brain, which cannot be easily achieved by traditional hydrogen therapy due to hydrogen’s low solubility and fast diffusion in brain inflammatory sites.

Synergy of photothermal and hydrogen therapy

The near - infrared (NIR) photothermal effect of PdH nanosheets can degrade amyloid - beta (Aβ) aggregates while transiently enhancing blood - brain barrier (BBB) permeability to improve drug delivery efficiency. This synergistic strategy of combining photothermal and hydrogen therapy offers a new approach for AD treatment, addressing multiple pathological features like Aβ aggregation, oxidative stress, and neuroinflammation.

Precise imaging - guided treatment

Photoacoustic imaging is utilized to monitor and guide the treatment process in real - time, enabling precise therapy. With NIR - II FL imaging technology, PdH nanosheets’ accumulation in the brain and stable use can be accurately observed, allowing for precise localization of AD lesions and treatment - outcome assessment, thus enhancing the treatment’s accuracy.

Efficient Aβ clearance and antioxidant therapy

Under NIR irradiation, PdH nanosheets can markedly reduce Aβ plaques and reactive oxygen species (ROS) levels in AD mice brains, shorten escape latency, and improve cognitive deficits. This provides robust evidence for AD treatment.

Significance

Problem or Issue

Alzheimer’s disease (AD), a prevalent neurodegenerative disorder, is marked by amyloid-beta (Aβ) accumulation, reactive oxygen species (ROS) production, and neuroinflammation. With the global population aging, the incidence of AD is rising yearly, projected to surpass 150 million dementia cases worldwide by 2050. AD poses a significant threat to global health and social care. However, no effective drug to reverse AD has been found yet due to its complex and unclear pathogenesis and long onset time.

What is Already Known

Hydrogen therapy shows promise due to its selective scavenging capacity of hydroxyl radicals (∙OH). But conventional hydrogen delivery methods fail to effectively accumulate hydrogen in the brain because of low solubility and rapid diffusion. Many therapeutic drugs for AD, such as peptides, organic molecules, and metal/supramolecular complexes, have been designed and synthesized, but they have average inhibitory efficiency and limited dissociation ability.

What this Paper Adds

This study reports ultra-small palladium hydride (PdH) nanosheets as a dual-functional nanomedicine for AD treatment. PdH nanosheets can release hydrogen efficiently through autocatalysis, enabling in situ ∙OH scavenging in the brain. Meanwhile, their near-infrared (NIR) photothermal effect can degrade Aβ aggregates and transiently enhance blood-brain barrier (BBB) permeability for improved drug delivery. In AD mice, intravenous PdH treatment combined with NIR irradiation significantly reduced escape latency in the Morris water maze, along with reductions in brain Aβ plaques and ROS levels. This study establishes a synergistic strategy of nanoscale hydrogen delivery and photothermal therapy, addressing the dual challenges of BBB penetration and multi-pathology intervention in AD.

Who would benefit from the new knowledge in this paper

The findings of this research will benefit patients with AD and their medical care teams. By offering a novel therapeutic strategy, it is expected to enhance the cognitive function, learning ability, and memory of AD patients. This provides new insights and experimental evidence for the development of more effective treatment methods for AD and also offers valuable reference for researchers in the field of nanomedicine, promoting the application of nanotechnology in the treatment of neurodegenerative diseases.

Introduction

Alzheimer’s disease (AD) is the most common neurodegenerative disease [1]. As the world’s aging population increases, AD incidence increases yearly [2]. It is estimated that by 2050, the number of patients with dementia worldwide will exceed 150 million [3]. Neurodegenerative diseases, such as AD, pose a huge threat to health and social care worldwide [4, 5]. However, no drug has been found to effectively reverse AD. The primary reasons include ADs complex and unclear pathogenesis and the long onset time. The main pathological feature of AD is abnormal amyloid-β (a-β42) accumulation [2, 6, 7]. Abnormal reactive oxygen species (ROS) production induced by α-β42 aggregates is also an important event in AD development [8, 9]. They are interrelated and interact with one another. Simultaneously, it is generally believed that AD is highly related to neuroinflammation [10, 2] [11], prompting the development of an anti-inflammatory strategy to overcome AD by eliminating oxidative stress and toxic free radical damage [9, 12, 13].

For decades, many therapeutic drugs, such as peptides, organic molecules, and metal/supramolecular complexes, have been designed and synthesized [14, 15]. A protein-capped (PC) cadmium sulfide metal nanoparticles (PC-CdS nanoparticles) have been constructed as a promising system for AD treatment [16]. Another recent study reported a new gold nanocluster modified with Cys-Arg dipeptide Au23(CR)14. This novel formulation was able to dissolve the exogenous Aβ mature fibrils completely to form monomers and the endogenous Aβ plaques in the AD brain [17]. However, many of them have average inhibitory efficiency and limited dissociation ability [5]. Therefore, it is necessary to develop high-quality nanomodulators with new structures and functions. Applications of palladium nanoparticles include different fields such as catalytic degradation, cancer Therapy, drug delivery, and hydrogen storage [18]. For example, EPP-AgPd1.5 NPs synthesized by Ruyu Li et al. has good photothermal conversion performance and anti-tumor ability. EPP-AgPd1.5 NPs have a great application prospect in bio-related detection and the treatment of malignant tumors in the future [19].

The biological effects of hydrogen molecules (H₂) are largely attributed to their ability to selectively scavenge highly reactive oxygen species (ROS), particularly the hydroxyl radical (∙OH), without interfering with physiologically important ROS involved in cellular signaling. The theoretical basis for hydrogen treatment of many inflammatory conditions [20]. Besides its good biomedical applicability, the high biosafety of hydrogen has been widely confirmed [19]. Therefore, hydrogen therapy can be used as a new strategy to treat AD. However, a problem with hydrogen therapy is that traditional hydrogen administration routes make it difficult for hydrogen to reach the inflammation site in the brain and effectively accumulate due to the low solubility and easy diffusion of hydrogen [5]. Therefore, achieving in-situ sustained release of hydrogen is crucial for improving the therapeutic effect of AD.

In this study, we designed small-sized palladium hydride (PdH) nanoparticles as hydrogen carriers and Pd-like autocatalysts to achieve in-situ sustained release of bioreducible hydrogen. PdH nanoparticles can release biologically reducing hydrogen via autocatalysis, selectively remove strongly oxidizing ∙OH in AD models, and reduce neuroinflammation. PdH nanoparticles degrade toxic amyloid aggregates via photothermal action, thereby reducing excessive Aβ production in AD mice and blocking AD progression. PdH treatment can significantly improve cognitive dysfunction in Balb/c mice.

Scheme 1.

PdH formation and its application in NIR-II FL-guided treatment of AD

Materials and methods

Chemicals

Palladium(II) acetylacetone (Pd(acac)2), poly(vinylpyrrolidone) (PVP, Mw = 30000), and NaBr were purchased from Aladdin (Shanghai), China. Sodium hydroxide (NaOH) and N,N-dimethylformamide (DMF) were acquired from the Sinopharm Chemical Reagent Company (Beijing, China). Acetone was obtained from Sigma-Aldrich. Deionized water was used in all experiments.

Pd nanosheet synthesis

Pd(acac)2 (50 mg), PVP (30 mg), and NaBr (52.2 mg) were dissolved in a mixture of DMF (10 mL) and H₂O (2 mL). The container was filled with CO to 1.0 bar and heated at 80 °C for 3 h with continuous stirring. After cooling to 0 °C, the obtained product was precipitated using 15 mL of acetone [12].

PdH nanoparticle synthesis

Pd nanoparticles were poured into a 20 mL vial, and the hydrogen generated by the hydrogen generator was blown into the Pd nanoparticle solution. After 15 min, hydrogenation was stopped, and the vial containing the PdH nanoparticles was stored in the dark for later use.

Characterization

The samples were scanned using a JEOL-2100F transmission electron microscope (TEM). UV-visible spectral analysis of the different samples was performed using a TU-1901 dual-beam spectrophotometer (PerkinElmer). The near-infrared (NIR) second-region fluorescence emission spectrum was excited using a fluorescence spectrometer (Suzhou NIR Optical Co., Ltd., China) with an 808 nm excitation laser source.

In vitro phototherapy effect of PdH

An infrared imaging camera (Fluke Ti400) was used to monitor the temperature changes of the sample solutions of different concentrations after irradiation with an 808 nm laser for 5 min. The photostability of PdH was evaluated using three heating and cooling cycles. In vivo PA imaging was performed at intervals using an in vivo NIR-II FL system (NIR-Optics) and a multispectral photoacoustic tomography system.

Animal model

Balb/c mice (both male and female mice were used without selectivity, 6–8 weeks old, 17–19 g) were purchased from Weitong Lihua Experimental Animal Technology Co.Ltd (Beijing, China).All animals were raised under standard housing conditions. The Aβ_25~35 peptide was used for the study. Before intracerebroventricular injection, the peptide (1 mg) was dissolved in PBS to a 1 mg/mL stock solution and aged at 37 °C for 7 days to promote aggregation. Then, the Aβ_25~35 aggregates were administered via stereotaxic injection into the right lateral ventricle of the mice. A volume of 10 µL of the prepared Aβ_25~35 aggregates solution was injected slowly into the right lateral ventricle of the mice via stereotaxic (Anteroposterior, −0.5 mm; Mediolateral, −1.0 mm; Dorsoventral, −2.5 mm).

Animal behavior experiment

This experiment utilized rodents’ innate fear of water to motivate them to find a platform and reach the shore. They learn to use the relationship between environmental markers and hidden platforms via multiple training sessions to judge the platform. Position. The Morris water maze was widely used to detect rodents’ vision-related spatial and working memory. Some modifications were made to the Morris water maze test to assess mice’s spatial learning and memory abilities. The mice were trained in a circular pool (diameter: 90 cm; height: 50 cm) containing water. The water depth was 30 cm, and the temperature was maintained at 24 ± 1 °C. The pool was divided into four quadrants using water maze software (EthoVision XT), and an invisible escape platform (9 cm in diameter) was located at a fixed spatial location in the northwest quadrant, 1 cm below the water surface. The experiment lasted for six days. The spatial learning task consisted of five training days. The mouse was gently released into the water in the southeast quadrant as the starting point. The mouse was allowed to find the hidden platform for 60 s. If the mice did not find the platform within 60 s, they were manually guided to it for 10 s. Mouse traces were monitored using a camera, and escape latency was recorded. After five days of task acquisition, the platform was removed, and a probe trial was conducted 24 h later to assess short-term memory consolidation. In the experiment, the mice were placed in the water in the northeast quadrant and allowed to swim freely in the pool for 120 s. The time the mice spent in the target quadrant (southwest quadrant) and the number of mice that crossed the previous platform location were recorded to assess their spatial memory. All trials were recorded using the standard Morris water maze (MWM) procedure.

Euthanasia and anesthesia methods

This study strictly adhered to laboratory animal ethics to ensure humane treatment of mice and minimize their suffering.

Anesthesia methods

We used isoflurane (CAS No.R510-22–10) inhalation anesthesia in the experiment. Isoflurane, a colorless and highly volatile liquid, offers rapid induction, stable anesthesia effects, and quick recovery. A dedicated inhalation anesthesia machine with oxygen as the carrier gas was used for administration. The induction concentration was 2–3%, and the maintenance concentration was 1.5–2%. It usually took 2 - 3 minutes to induce anesthesia in mice.

Euthanasia methods

At the end of the experiment, mice were euthanized via sodium pentobarbital injection. The euthanasia dose was three times the anesthetic dose. To alleviate discomfort, local anesthetics and anticonvulsants were used together. All procedures were carried out in a professional laboratory facility by experienced personnel in line with the ethics committee - approved protocol.

Results and discussion

Palladium has a high hydrogen storage capacity because hydrogen atoms can be incorporated into its crystal lattice and is highly active in catalytic hydrogenation, especially at the nanoscale [21, 22, 23]. With these advantages, we developed PdH nanoparticles to deliver and release active hydrogen to reduce the inflammatory response in AD. TEM was used to characterize the morphology and size of the PdH nanoparticles (Fig.s 1a-b). Delivering therapeutic substances to the brain is a major challenge in treating neurological diseases [24, 25]. This is mainly due to the blood-brain barrier (BBB) restricting the entry of drugs [2, 26, 27] and the limited stability and distribution of drugs in brain tissue. PdH nanoparticles exhibit a hexagonal nanosheet morphology of Pd, are small and uniform (approximately 37 nm in diameter), and have good dispersion, which is beneficial for storing, transferring, and releasing hydrogen and can achieve efficient BBB delivery [15]. The crystal structure nature of PdH was analyzed by X-ray diffraction (XRD) pattern. As shown in Fig. 1c, it could be found that the PdH leads to a slight shift of diffraction peaks towards small angle compared to Pd [28]. According to Bragg’s law, this shift can be attributed to lattice expansion, which is typically associated with the incorporation of hydrogen atoms into the interstitial sites of the Pd lattice to form PdH.

Fig. 2.

(a) photothermal curves and images of PdH solutions with different concentrations under 808 nm (1 W cm⁻²) irradiation. (b) PdHH (0.5 mg mL⁻¹) during 3 laser on/off cycles (808 nm, (1 W cm⁻²). (c) Photothermal conversion efficiency of PdH

Fig. 1.

(a) Electron microscope image of PdH nanoparticles. (b) TEM particle size of PdH nanoparticles. (c) XRD patterns of PdH nanoparticles

Taking advantage of the superior NIR absorption ability of PdH, the photothermal properties of PdH were tested under 808 nm irradiation. As expected, the temperature of the PdH solution increased sharply with increasing time and concentration (Fig. 2a). After three heating and cooling cycles, no obvious attenuation was detected during heating, indicating the excellent photothermal stability of PdH (Fig. 2b). Based on the fitting cooling curve in Fig. 2c, the photothermal conversion efficiency (η) of the PdH was calculated at 38.50%, suggesting that the PdH have the potential to function as a competitive photothermal agent. Hydrogenated palladium nanomaterials showed to have high near-infrared photothermal conversion efficiency and good biocompatibility, and can serve as excellent photothermal therapy (PTT) and photoacoustic imaging (PAI) agents.

The UV-visible-NIR spectra of Pd and PdH exhibit similar broad absorptions in the range of 500 ~ 800 nm, which is the typical absorbance of Pd (Fig. 3a). In the subnanometer layer, PdH exhibits strong light absorption in the NIR region while possessing superior optical properties [15, 21]. Additionally, the biological reduction ability of PdH nanoparticles to release hydrogen was studied using the redox probe methylene blue (MB). Generally, MB must be combined with Pt nanoparticles as catalysts for hydrogen detection [29]. Fortunately, PdH nanoparticles can act as a Pt-like catalyst (called an autocatalyst), assisting the hydrogenation reaction (such as assisting MB in detecting hydrogen), presenting that the hydrogen released by PdH has a higher reduction rate. According to the mechanism of MB detecting hydrogen [30], indicates a decrease in the amount of MB. While PdH catalyzed a rapid decrease in the MB absorbance, the pure Pd NPs showed negligible change over the same reaction period (Fig. 3b–c). This control experiment confirmed that the mere presence of Pd is not sufficient to drive the reaction. The hydrogenation of MB was specifically facilitated by PdH, which acts as an active hydrogen carrier and release agent. This comparison powerfully underscored the critical role of the formed PdH in enabling and accelerating H₂ release and subsequent MB reduction. The reducing capacity of PdH and PdH with laser irradiation was further evaluated by comparing the changes in absorption values of MB at 665 nm (Fig. 3d).This indicated that the temperature of PdH increases after being irradiated by the laser, which further enhances the reduction of PdH, further validating the prospect of nanoparticles being more attractive in the NIR window. The ABTS assay (Fig. 3e) confirmed its efficient neutralization of ABTS⁺ radicals, evidenced by the progressive decolorization of the solution. This was quantitatively paralleled by the DPPH assay (Fig. 3f), which showed a similar concentration-responsive scavenging profile, visually marked by the characteristic color transition from purple to yellow. These experiments validate that PdH functions as an effective free-radical scavenger, firmly supporting our fundamental hypothesis that its therapeutic benefits in alleviating AD pathology are rooted in its robust antioxidant activity.

Fig. 3.

(a) UV spectra of Pd and PdH nanoparticles. (b) Detection of reducing hydrogen using MB, UV spectrum of MB solution treated with Pd nanoparticles. (c) Detection of reducing hydrogen using MB, UV spectrum of MB solution treated with PdH nanoparticles. (d) UV spectra of PdH and PdH solutions of MB solutions treated under laser irradiation. (e) Absorbance change of ABTS at 736 nm and corresponding digital photos of PdH at different concentrations. (f) Absorbance change of DPPH at 520 nm and corresponding digital photos of PdH at different concentrations

Mice anesthetized before drug injection were used as controls (pre) (Fig. 4a). The photoacoustic signals in the NIR first and second regions increased rapidly from NIR-I to NIR-II by with tail vein injection of PdH nanoparticles. NIR-II is more precise in its positioning in the brain than a laser and has good results in AD and treatment guidance. NIR photothermal imaging results show that PdH nanoparticles can be effectively accumulated and stably used in the brain. PdH-ICG 2 h developed in the brain (Fig. 4b), indicating it readily crosses the BBB. The main obstacle in treating brain diseases is the body’s own BBB [27]. Small-sized PdH plays an important role in entering the inflammatory site of the brain to treat AD.

Fig. 4.

(a) PdH-treated NIR-I and NIR-II photoacoustic imaging of the brain. (b) NIR-II FL images of AD mouse at different time points after intravenous injection of PdH-ICG

Fig. 5.

The behavioral evaluation of PdH-treated Balb/c mice under NIR laser (808 nm) irradiation (a) time schedule of model construction, i.V. injection and therapeutic evaluation. (b) Escape latency of mice irradiated with WT, AD, PdH alone, and PdH combined with NIR laser. (c) Representative swimming trajectories of mice in the WT group, AD group, PdH group and PdH combined with 808 nm laser irradiation group. (d) Representative heat map of y-maze test. (e)y maze experiments were spontaneously alternated. Data are expressed as mean ± SD, n = 4 per group,WT group mouse number 48,49,45,90, AD group mouse number 59,54,51,1, PdH treatment group mouse number 46,52,57,42, PdH+Laser treatment group mouse number 58,56,47,53

Alzheimer’s disease is a neurodegenerative disorder characterized by progressive decline in cognitive function, especially short-term memory [31]. Inorganic nanoparticles are highly stable, small in size, easy to prepare and have excellent permeability. Small inorganic nanoparticles ( < 100 nm) can effectively and effortlessly penetrate a wide range of tissues, including the blood-brain barrier [32]. Inspired by its significant neuroprotective effect and BBB penetration ability, we next evaluated the therapeutic effects of PdH in Aβ-AD mice (Fig. 5a). The Aβ-AD mice were randomly grouped and administered different treatment. Wild-type mice (named as WT) receiving saline served as healthy controls. Mice in PdH and PdH+Laser groups received intravenous injections of PdH (5 mg/kg) every 3 days for 4 weeks. For the PdH+Laser group, each injection was followed by NIR laser irradiation (808 nm, 1 W/cm²) at the skull surface for 5 min to trigger the release of H₂. Upon completion of the 4 weeks treatment, the comprehensive behavioral battery (MWM and Y-maze) was conducted to evaluate the functional recovery of cognitive abilities.

As illustrated in Fig. 5b, all groups exhibited a progressive shortening of escape latency throughout the 6 days spatial training in the Morris water maze (MWM), indicating a general learning curve. However, the Aβ-AD mice consistently demonstrated significantly prolonged escape latencies and path lengths to locate the hidden platform compared to the WT group. As the training progressed, the performance gap between the AD and WT groups widened, recapitulating the profound spatial learning and memory deficits characteristic of AD pathophysiology. Notably, our therapeutic intervention markedly ameliorated these deficits. PdH administration alone shortened the escape latency, suggesting a beneficial effect. Most importantly, the PdH+Laser group exhibited a performance that was comparable to the WT group, underscoring a near-complete restoration of spatial learning ability. This pronounced effect under NIR laser can be attributed to the on-demand and enhanced release of H₂ from the PdH, driven by the photothermal effect, which potentiated the therapeutic outcome. Corroborating this, the probe test on day 6 revealed stark differences in search strategies (Fig. 5c). Mice in the AD group displayed predominantly aimless, thigmotactic swimming patterns, whereas the swimming trajectories of the PdH+Laser treated mice were highly focused and predominantly confined to the target quadrant where the platform was previously located, indicating precise spatial memory. To further validate the cognitive improvements, we employed the Y-maze test to assess short-term spatial working memory. Consistent with the MWM results, spontaneous alternation was significantly impaired in the AD group compared to the WT group. This deficit was partially rescued by PdH treatment alone and more robustly reversed by PdH+Laser, bringing the alternation percentage to a level akin to that of normal mice (Fig. 5d).

Collectively, these behavioral findings compellingly demonstrate that PdH-based therapy, especially when potentiated by NIR laser irradiation, effectively mitigates cognitive and learning deficits in Aβ-AD mice. We postulate that the behavioral recovery is intrinsically linked to the multifaceted therapeutic mechanisms of PdH. The nanoparticle enables sustained and NIR-triggered release of H₂, which directly scavenges cytotoxic •OH radicals, thereby alleviating oxidative stress and subsequent neuroinflammation—key drivers of synaptic dysfunction and memory loss in AD. Furthermore, the potent NIR-II photothermal effect of PdH not only facilitates H₂ release but may also contribute to the suppression of Aβ aggregation. The integration of PA imaging allows for precise guidance, ensuring the therapeutic agents are delivered to the target sites. Therefore, the restoration of spatial learning and memory behaviors observed in our study can be reasonably attributed to this synergistic anti-oxidative and anti-inflammatory action within the brain.

Conclusion

The excellent small-sized therapeutic nanoparticles we designed and synthesised can easily cross the blood-brain barrier to the site of inflammation in the brain and achieve in situ release of hydrogen to reduce neuroinflammation. Meanwhile, pdH shows good photothermal properties under near-infrared light irradiation, which enhances the therapeutic effect of pdH on AD. Most importantly, the treatment with PdH under NIR-II irradiation significantly improved cognitive function, learning, and memory abilities in AD mice, as robustly demonstrated by our behavioral tests. We found that PdH nanoparticles can degrade amyloid aggregates via photothermal action by treating AD model mice, reduce the excessive Aβ production in the AD mice brains, and block AD progress. Moreover, PdH and photothermal treatment significantly improved cognitive function, learning, and memory abilities in AD mice. This study’s proposed pdH nanomedicine strategy may open a new window for treating AD.

Author contributions

LJY and MXZ were responsible for literature searches, study design, data analysis interpretation, and drafting the initial manuscript. WJZ, ZGL and KQZ organized and revised the manuscript according to submission guidelines. HLL and YJQ participated in data management. XP polished the language to enhance clarity. ZLZ and WWZ provided guidance on research methods, supervised data analysis, and helped interpret the results. All authors reviewed and approved the final manuscript.

Funding

This study was supported by the Shanxi Province Science and Technology Achievement Transformation Guidance Special Project [#202404021301058 to WZ] and the Fundamental Research Program of Shanxi Province [#202203021221243 to WZ].

Data availability

The data that support the findings of this study are available from authors upon reasonable request.

Declarations

Ethical approval

All animal procedures were performed per the standards of the Chinese National Regulations for the Care and Use of Laboratory Animals. The animal protocol was approved by the Institutional Animal Use and Care Committee of Shanxi Medical University (Approval No. 2016LL141, Taiyuan, China).

Consent for publication

All authors have read and approved the final manuscript.

Competing interests

The authors declare no conflict of interest