Cortical Circuitry and Synaptic Dysfunctions in Alzheimer’s Disease and Other DementiasView this Special Issue
Bushen-Tiansui Formula Improves Cognitive Functions in an Aβ1–42 Fibril-Infused Rat Model of Alzheimer’s Disease
Bushen-Tiansui Formula (BTF) was empirically updated from a classical prescription named Kong-Sheng-Zhen-Zhong pill. It is based on the traditional Chinese medicine theory of the mutual relationship between the brain and the kidney and is intended to treat neurodegenerative diseases. This formulation has been used for several years to treat patients with Alzheimer’s disease- (AD-) like symptoms in our clinical department. However, the medicinal ingredients and the mechanisms by which BTF improves cognition and memory functions have not been characterized. In this study, we used UPLC-MS to generate a chromatographic fingerprinting of BTF and identified five possible active ingredients, including stilbene glycoside; epimedin A1, B, and C; and icariin. We also showed that oral administration of BTF reversed the cognitive defects in an Aβ1–42 fibril-infused rat model of AD, protected synaptic ultrastructure in the CA1 region, and restored the expression of BDNF, synaptotagmin (Syt), and PSD95. These effects likely occurred through the BDNF-activated receptor tyrosine kinase B (TrkB)/Akt/CREB signaling pathway. Furthermore, BTF exhibited no short-term or chronic toxicity in rats. Together, these results provided a scientific support for the clinical use of BTF to improve learning and memory in patients with AD.
Alzheimer’s disease (AD) is the leading cause of dementia worldwide, but the etiology and pathogenesis of this disease have not been characterized. Accumulation of β-amyloid peptide (Aβ) in the brain and hyperphosphorylation and cleavage of the microtubule-associated protein Tau are hallmarks of AD . However, over the last decade, a series of new drugs designed to clear neurofibrillary tangles have failed to improve or reverse AD, which indicated that neurofibrillary tangles correlated weakly with the degree of dementia in patients with AD . In contrast, synaptic loss has been strongly correlated with cognitive impairment, and it may be the pathological basis of cognitive changes in AD .
Neurotrophins are growth factors that regulate neuronal development, differentiation, and survival. Brain-derived neurotrophic factor (BDNF) is an important neurotrophin that is distributed extensively throughout the central nervous system. BDNF binds to receptor tyrosine kinase B (TrkB) and triggers activation of the downstream TrkB/Akt and TrkB/CREB signaling pathways, resulting in the synthesis of synaptotagmin (Syt) and PSD95 in synapses. Synaptotagmin and PSD95 confer protection by regulating the repair of synapses and the reconstruction of neural circuits to improve learning and memory in animals with dementia [4–6]. Studies have shown that the expression of BDNF was reduced in the brains of patients with AD with synaptic loss . A strategy of using BDNF as a therapeutic agent for neurologic disorders was carried out based on this preclinical evidence. Unfortunately, the outcomes of several clinical trials involved the intrathecal infusion of recombinant BDNF to treat patients with amyotrophic lateral sclerosis have been disappointing due to the short in vivo half-life and poor delivery of BDNF [8, 9]. Thus, novel strategies to directly stimulate production and expression of BDNF by exploring drugs may result in better therapeutic outcomes.
Facing the treatment complexity of AD, a growing body of reports have suggested that traditional Chinese medical formulas (TCMFs) with multitarget effects may result in improved cognitive function [10–12]. Bushen-Tiansui Formula (BTF) is derived from a classic prescription, Kong-Sheng-Zhen-Zhong pill (Qianjin Formulas), and was empirically modified from this classic prescription by adjusting the composition and proportion of herbs. Its formula is intended to meet neurodegenerative diseases and embodies the Chinese medicine theory of the mutual relationships between the brain and the kidney, in which the core statements are that the deficiency of kidney function leads to the encephalon loss, and the kidney gives birth to the encephalon and the brain stores marrow . BTF has been utilized for several years to treat patients with AD-like symptoms in our clinical department. Nonetheless, the pharmacological ingredients in BTF, and the mechanisms by which they improve cognitive and memory functions, have not been characterized. Our previous study reported that icariin, a major active component from Herba Epimedium brevicornum (Yin-Yang-Kuo) that belongs one of the herbs in BTF, improved synaptic plasticity in an Aβ1–42 rat model of AD . As a single compound, however, it will be a long-term task to develop it into an innovative promising drug for clinical use.
In this present study, we characterized the formula composition of BTF and generated a chromatographic fingerprint profile. We also evaluated the effects of BTF on cognition and memory functions in a rat model of AD and evaluated BFT-induced expression of BDNF and the activation of the TrkB/Akt/CREB cascaded signaling pathways. Our study provided scientific support for the clinical use of BTF to improve learning and memory in patients with AD.
2. Materials and Methods
2.1. Preparation of BTF Extract
BTF is comprised of six herbs mixed in the proportions summarized in Figure 1(a). All the mentioned botanical names can be checked following the database of http://www.theplantlist.org. The herbal names and Chinese names were retrieved from the 2015 edition of the Chinese pharmacopeia. All herbs in BTF were purchased from the TCM pharmacy of the Second Xiangya Hospital, Central South University (CSU), Changsha, Hunan Province. Voucher specimens (201605301-6) were well deposited at the department of integrated traditional Chinese and Western medicine at the Second Xiangya Hospital, CSU. The herbs were soaked in a 10 times volume of ddH2O () for 1 h and then boiled twice for 1 h each. The two boiled solutions were combined and concentrated under vacuum and then freeze-dried to yield a lyophilized powder (output rate of 14.3% ()). The lyophilized powder of BTF was stored at -20°C until used.
2.2. Chromatographic Fingerprint Analysis of BTF Extract
Ultrahigh-performance liquid chromatography-tandem mass spectrometer (UPLC-MS) was utilized to analyze the principal components in BTF. Standard compounds including stilbene glycoside (BWB50367), epimedin A1 (BWB50192), epimedin B (ASB-5159-010), epimedin C (ASB-5160-010), and icariin (GBW09541) were purchased from the National Standard Center. A CNW Athena C18-WP column (, 5 μm) was used as a solid phase and maintained at 35°C while the spectrum analysis was performed. The mixture of water (A) and CH3CN (B) was acted as a mobile phase with a gradient elution ratio as follows: 0-10 min 20%-30% B, 10-22 min 30% B, 22-25 min 30-33% B, and 25-30 min 33-80% B. The monitoring wavelength was 270 nm with a flow rate of 0.5 mL/min. Electrospray ionization (ESI) mode was used for mass spectrometry with a capillary voltage of 3500 V.
2.3. Aβ1-42 Preformed Fibril Preparation
Aβ1–42 (Sigma, USA) was dissolved at 1 mg/mL in hexafluoroisopropanol (HFIP) at room temperature and then sonicated in a bath sonicator for 5 min. The HFIP was evaporated using a gentle stream of nitrogen gas, and then nine volumes of ice-cold distilled water were added while vortexed occasionally. Keeping the solution on ice for 30 min, one volume of 10x fibril-forming buffer (0.2 M NaPi, 1.5 M NaCl, 0.2% NaN3, pH 7.5) was added and vortexed repetitively. We sealed the solution tube and stored at 37°C for one week and vortexed daily. Fibril formation was verified using a Thioflavin T binding assay according to a previous report . The Aβ fibrils were stored at -80°C.
2.4. Aβ1–42 Fibril-Infused Rat Model and BTF Treatment
The Aβ1–42 fibril-infused rat model was established as described in our previous study . In brief, adult male Sprague-Dawley (SD) rats (weight: 200−220 g) were anesthetized with isoflurane and then fixed in a stereotactic apparatus. The Aβ1–42 fibrils (3 μL) were delivered bilaterally into the lateral ventricles at a rate of 0.5 μL/min (from bregma, anteroposterior (AP) −1.0 mm, 1.5 mm lateral to the sagittal suture, and 4.6 mm beneath the dura). An equivalent volume of sterile saline was injected as a sham group (). Following infusion for 3 days, rats that received the Aβ1–42 infusion were randomly distributed to two groups ( for each group). According to clinical use dose, the test group was orally administered 27 g/kg BTF, and another group was orally administered an equivalent volume of sterile saline. The animals were dosed once per day for 28 days. The rats were assigned to gender- and age-matched treatment groups using a randomized block design. The total experimental period is summarized in Figure 2(a), and the experimental procedures were approved by the Review Committee of Central South University (Changsha, China).
2.5. Morris Water Maze Test
The Morris water maze test was carried out from 28 days after the beginning of BTF intervention. Rats in three groups were trained in a round, diluted ink water-filled tub () in an environment rich with extra maze cues. An invisible escape platform () was located in a fixed spatial location 1 cm below the water surface independent to utilize extra maze cues to determine the location of the platform. At the beginning of each trial, the rats were placed in the water maze with their paws touching the wall from one of four different starting positions (N, S, E, and W). Each rat was subjected to four trials per day for five consecutive days with a 15-minute intertrial interval. The maximum trial length was 60 s, and the rats were manually guided to if they did not reach the platform in the allotted time. Upon reaching the invisible escape platform, the rats were left on it for an additional 10 s to allow for a survey of spatial cues in the environment to guide future navigation to the platform. The temperature of the water was monitored every hour and maintained between 22 and 25°C. Following the 5 days of task acquisition period, a probe trial was presented when the platform was removed, and the number of platform crossings and the percentage of time spent in the quadrant that previously contained the escape platform during task acquisition was recorded over 90 s. The whole trial process and the analysis of behavioral parameters were recorded through the ANY-maze video tracking system (Stoelting Co., USA).
2.6. Electron Microscopy
Synaptic ultrastructure detection was determined by electron microscopy as described previously . Briefly, after deep anesthesia, rats were perfused transcardially with 4% paraformaldehyde in PBS. Hippocampal slices were postfixed in cold 2.5% glutaraldehyde, then dehydrated, soaked, and embedded through a graded acetone series. The embedded sections were dual-stained with uranyl acetate and lead citrate and visualized at 100 kV in a transmission electron microscope (Hitachi Ltd., Tokyo, Japan). Synapses were evaluated by the presence of synaptic vesicles and postsynaptic density, including the number of synapses, the width of each synaptic cleft, the thickness of the postsynaptic density, and the length of the synaptic active zone.
2.7. Western Blotting
Western blotting was performed using a standard protocol. Rat hippocampus tissue was sonicated and lysed with RIPA lysis buffer, and insoluble pellets were removed by centrifugation at 15,000 × g for 15 min at 4°C. Protein concentration was measured using the BCA method, and the lysates were stored at -80°C until analysis. Equal amounts of protein (30-40 μg) were loaded for blotting with anti-p-TrkB/TrkB (1 : 1000, #sc-8058/#sc-7268, Santa Cruz Biotechnology, CA, USA), anti-p-Akt/Akt (1 : 1000, #4060/#9272), anti-p-CREB/CREB (1 : 500, #9189/#9197), anti-Syt (1 : 1000, #14558), and anti-PSD-95 (1 : 1000, #2507) (Cell Signaling Technology, Denver, MA, USA), and anti-BDNF (1 : 500, #108319, Abcam, Cambridge, UK).
Immunohistochemistry (IHC) was performed to visualize BDNF and p-Akt according to the manufacturer’s instructions (Invitrogen). Briefly, free-floating 25 μm thick serial hippocampus sections were treated with 0.3% hydrogen peroxide for 10 min, and then, sections were rinsed three times with PBS and blocked in Reagent 1A for 10 min followed by incubation with BDNF (1 : 300) or p-Akt (1 : 500) antibody at 4°C overnight. After PBS washing, sections incubated with a biotinylated second antibody Reagent 1b followed by the conjugate enzyme Reagent 2 for each 10 min. Finally, a chromogen AEC single solution was utilized to develop the signals and captured in a microscope (BX51TF, Olympus, Tokyo, Japan) with cellSens standard V3 detection system.
2.9. Hematoxylin and Eosin Staining
Multiple organs were collected and were immediately fixed in 4% formaldehyde. After immersion, organs were dehydrated by gradual soaking in alcohol and xylene, embedded in paraffin, and then sliced into 5 μm thick sections, which were stained with standard hematoxylin and eosin (H&E) staining protocol . Sections were visualized under a digital optical microscope (Olympus, Tokyo, Japan).
2.10. Statistical Analysis
Statistical analysis was performed with Prism 7.0 (GraphPad software). All data were expressed as . from three or more independent experiments. Histological data were analyzed by one-way ANOVA. The threshold for significance for all experiments was set at , and smaller values were represented as and #.
3.1. Chromatographic Fingerprinting Analysis of BTF
The pharmacological effect of traditional Chinese medicine formulas (TCMFs) is derived from combinations of active compounds. To investigate the possible major medicinal compounds in BTF, a qualitative assessment of ingredients was tentatively characterized the by UPLC-MS system and the chromatographic fingerprint was established as illustrated in Figure 1(b). Approximately 14 chromatographic peaks can be defined in the characteristic profile of BTF. According to the ratio in the MS detection, five of these peaks (peak 1-5) were identified as stilbene glycoside (peak 1, 405.1216 [M-H]-), epimedin A1 (peak 2, 837.5901 [M-H]-), epimedin B (peak 3, 807.2715 [M-H]-), epimedin C (peak 4, 821.2855 [M-H]-), and icariin (peak 5, 677.2433 [M+H]+) (Figure 1(b)). These compounds were chemical components of Radix Polygoni Multiflori Preparata and Herba Epimedii Brevicornus based on the previous phytochemistry studies [18, 19]. Further, standard substances for these five compounds were purchased and their mixed solution was subjected to UPLC-MS analysis with the same elution conditions. The comparison of chromatograms showed similar UV absorption spectra and retention times for each peak. Therefore, these data suggested that stilbene glycoside; epimedin A1, B, and C; and icariin were characteristic components in BTF.
3.2. Oral Administration of BTF Rescues Cognitive Deficits in Aβ1–42 Fibril-Infused Rats
To evaluate the effects of BTF on cognitive function in the AD model, hippocampus-dependent spatial memory in Aβ1–42 fibril-infused rats was assessed using the Morris water maze test. The average escape latency to the hidden platform for each of the five acquisition days was calculated and plotted (Figures 2(b) and 2(c)). Two-way mixed ANOVA () for latency revealed a main effect of the training day () and of the group (), but there was no interaction (Figure 2(b)). The AUC of the escape latency was significantly greater in the saline-treated Aβ1–42 fibril-infused rats compared with that in the sham group, which indicated impaired acquisition of the spatial learning following intracerebroventricular injection of Aβ1–42 fibrils. Memory recall for the platform location was assessed in the probe trial by removing the platform and allowing the rats to search for 90 s. Compared with the sham group, saline-treated Aβ1–42 fibril-infused rats spent a significantly lower percentage of their time and fewer platform site crossing in the quadrant that formerly contained the hidden platform, which was indicative of severe deficits in spatial memory recall. Compared with the sham group rats, the Aβ1–42 fibril-infused rats treated with BTF spent a significantly greater percentage of time in the target quadrant and crossed the target quadrant more frequently (Figures 2(d)–2(f)), which demonstrated the rescue of spatial memory.
3.3. Oral Administration of BTF Prevents Synaptic Loss in Aβ1–42 Fibril-Infused Rats
Synaptic loss and decreased hippocampal synaptic plasticity are believed to be the basis of cognitive impairment in the early phases of AD . We directly quantified the synaptic density and evaluatedsynaptic ultrastructure parameters in the CA1 region of the hippocampus using electron microscopy (EM). Saline-treated Aβ1–42 fibril-infused rats showed significantly reduced synaptic density, perforated synapses, synaptic active zones length, and postsynaptic density thickness and increased synaptic cleft width. The oral administration of BTF significantly reversed these ultrastructure changes but did not alter the curvature of the synaptic interface (Figures 3(c)–3(g)). To confirm these findings, we performed immunoblotting for the presynaptic marker synaptotagmin and the postsynaptic marker PSD95. Saline-treated Aβ1–42 fibril-infused rats displayed the considerably decreased expression of synaptotagmin and PSD95, which was indicative of synaptic degeneration in this AD model. Treatment with BTF reversed Aβ1–42 fibril-induced reduction of synaptic marker expression (Figure 3(h)). These results indicated that oral administration of BTF inhibited synaptic loss and improved synaptic plasticity in Aβ1–42 fibril-infused rats.
3.4. BTF Promotes BDNF Expression and Activates Downstream Signaling Pathways in the Rat Brain
To explore the possible mechanisms by which BTF improved cognitive function in Aβ1–42 fibril-infused rats, we investigated BDNF and its downstream signaling pathways. Following behavioral testing, we monitored BDNF expression in rat brains by immunoblotting analysis using an anti-BDNF antibody. Surprisingly, quantitative analysis revealed that BDNF was regained to normal levels in BTF-treated Aβ1–42 fibril-infused rats. Furthermore, the expression of phosphorylated TrkB, but not total TrkB, was notably elevated following BTF treatment (Figure 4(a), 1-3th panels). As expected, the main proteins on TrkB signaling pathway were more prominently phosphorylated in Aβ1–42 fibril-infused rats treated with BTF than in those treated with saline, as were the downstream activation of Akt/CREB signaling cascades (Figure 4(a)), which resulted in the synthesis of PSD95 in synapses. These results were confirmed by immunohistochemistry (IHC) staining of rat hippocampi using anti-BDNF (Figure 4(b)) and p-Akt S473 (Figure 4(c)). Therefore, these data indicated that the promotion of BDNF expression by BTF treatment led to the activation of its downstream TrkB-Akt/CREB signaling cascades might be responsible for the cognitive function improvement in AD rats.
3.5. Oral Administration of BTF Presents No Toxicity for Rats
Drug safety is an important consideration in clinical investigations. As a compulsory experiment, we performed a 12-week chronic BTF toxicity study in Sprague-Dawley (SD) rats (200–220 g) with a daily dose of 54 g/kg. Continuous weekly weight records and the H&E staining of tissue sections from multiple organs (the heart, liver, spleen, lung, kidney, testis, and ovary) showed that there were no significant differences between rats administered with BTF and those administered with saline (Figures 5(a) and 5(b)). Besides, blood levels of RBC, HB, WBC, and ESR were within the normal ranges in rats that received BTF (data not shown). Thus, this study supports that the oral administration of BTF is safe and trustworthy for treating AD.
Traditional Chinese medical formulas (TCMFs), developed based on the theory of the holistic body in traditional Chinese medicine (TCM), have been historically proven to be effective drugs in treating human diseases. Kidney-brain communication and reciprocity is one of the most important theories in TCM. This theory states that the kidney is a producer of the encephalon, and the cerebral marrow will be sufficient if the spirit in the kidney is exuberant . Modern medical epidemiological data has shown that individuals at all stages of chronic kidney disease (CKD) are at higher risk of developing cognitive disorders and dementia . Studies have proven that vascular injury, endothelial dysfunction, and direct neuronal toxicity may be potential factors in the pathophysiologic link of the kidney-brain axis [22–24]. “Kong-Sheng-Zhen-Zhong” pill was detailed in the classical medical book “Qianjin Formulas” written by Sun Simiao, a famous ancient Chinese medical expert. This formula is comprised of many nourishing herbs to achieve the treatment objectives that replenish vital essence, tonify kidney yin, and nourish the bone marrow . Bushen-Tiansui Formula is an empirically improved version of the Kong-Sheng-Zhen-Zhon pill developed in our department by adjusting the composition and proportion of herbs, and the development of the application of BTF in neurological disorders was based on the theory of the kidney-brain axis. It has been shown to effectively treat neurodegenerative diseases, including AD. Specifically, BTF has been proven efficient in practice in our clinical department for improving cognitive and memory functions in patients with AD. Our discoveries about the development of a chromatographic fingerprint for BTF through the modern analytical techniques, and characterization of the mechanisms by which BTF improved cognition provided a scientific basis for expanded use of this formula.
UPLC-MS combines the efficient separation capabilities of UPLC and the great power in the structural characterization of MS and provides a new powerful approach to identify the constituents in TCMFs rapidly and accurately . In addition, diode array detection (DAD) is a commonly used detection technique for HPLC analysis. The combined use of DAD and MS can provide excellent specificity by providing orthogonal information for each peak. Fragmentation can be used to identify compounds using databases, and novel compounds can be identified using data deconvolution software sand spectral matching. In our current study, five components were identified that were associated with two herbs in BTF (Figure 1(b)). According to phytochemistry studies [27, 28], the main components of Gui-Ban and Long-Gu were amino acids with no conjugated bonds and minerals, respectively, which do not typically contain chromophores and cannot be detected by UV analysis. Moreover, the monitoring wavelength of 270 nm may not have captured the absorbance of asarone and saponins present in Shi-Chang-Pu and Yuan-Zhi, respectively. Therefore, UV detection may not be sufficient to characterize BTF [29, 30]. Furthermore, the lack of commercial standards for analysis and characterization may also be a limiting factor. In terms of the brain availability of the molecules we identified by UPLC-MS, those compounds belong to polyphenols (PPs). PP metabolites could indirectly regulate the cerebrovascular system or directly act as neurotransmitters crossing the blood-brain barrier, while the gut microbiota plays a crucial role in metabolizing dietary polyphenols into lipid-soluble metabolites that are absorbed by cells [31, 32]. In our experiment, we administrated BTF by oral gavage and believed that the metabolites of PPs that were transformed from the gut microbiota or live metabolism could pass through the blood-brain barrier (BBB) and accumulated in the brain.
A pathological hallmark of AD is the presence of amyloid plaques within the brain, an observation that led to the β-amyloid (Aβ) cascade hypothesis of AD [33, 34]. Attention has since focused on the isoforms and their physiological function, specially Aβ1–42. Recent studies have shown that AD may be caused by Aβ aggregates that adopt alternative conformations, resulting in prion-like self-propagation [35–37]. Intracerebroventricular injection of Aβ1–42 fibrils has been shown to induce hyperphosphorylation of tau, tangle formation, and leading eventually to neuronal death and dementia and has also been reported as seeds to induce endogenous Aβ aggregation then trigger neurotoxicity [33, 38]. These findings resulted in the development of models using endogenous Aβ and other plaque-associated factors without the need to overexpression of potentially confounding amyloid precursor protein (APP) domains, which naturally became a classic and popular AD animal model. Even so, further investigation on transgenic models of AD will be scheduled to fully assess the potential benefit of BTF and to exclude any side effect caused by impairment due to mechanical injury in the Aβ1–42 fibril-infused rats used in our studies.
BDNF exerts its biological functions on neurons through two transmembrane receptors: TrkB and p75 neurotrophin receptor (p75NTR) . TrkB is a high-affinity catalytic receptor for several neurotrophins and is highly enriched in the hippocampus . Phosphorylation of TrkB following stimulation by BDNF triggers the downstream activation of the PI3K/Akt/CREB signaling cascades, driving synaptotagmin and PSD95 synthesizing in endoplasmic reticulum (ER) and trafficking to synapses throughout the neurons, which results in rapid and dendrite-wide sensitization for synaptic potentiation [41, 42]. Intriguingly, BDNF gene transcription is controlled by the CREB family of transcription factors, and PSD95 interacts with TrkB receptor for BDNF [43, 44]. These two circulation pathways promote the high expression of BDNF and PSD95, as well as stable TrkB receptors for responding to BDNF stimulation effect. As a result, the activation of these pathways results in a positive feedback loop in which synapses become more responsive to BDNF, which leads to increased transport of PSD95 to synapses. Together, we noted that BTF significantly stimulated the expression of BDNF and subsequently induced the activation of the TrkB-Akt/CREB cascaded signaling pathways, which resulted in increased synthesis of downstream synaptotagmin and PSD95 in synapses. These results identified a potential mechanism of BTF-induced significant stabilization of synaptic plasticity in Aβ1–42 fibril-infused rats.
In conclusion, our study generated a chromatographic fingerprint for the Bushen-Tiansui Formula (BTF) that has been utilizing in our clinic department for several years and further confirmed that BTF improved cognitive function through the prevention of synaptic loss, as demonstrated in the classical AD model of Aβ1–42 fibril-infused rats. Furthermore, the mechanism investigation revealed that BTF stimulated the expression of BDNF and triggered the activation of the TrkB/Akt and TrkB/CREB pathways, which resulted in the upregulation of synaptotagmin and PSD95 expression in synapses. In short, this study provides a compelling scientific basis for expanding the application of BTF in the clinic, and the ongoing randomized clinical trial of BTF, in turn, will confirm the reliability of our reporting, which provides a reliable pathway for the treatment of AD.
The data used to support the findings of this study are included within the article.
Conflicts of Interest
The authors declare that they have no conflict of interest.
S.L. conceived the project, designed the experiments, analyzed the data, and wrote the manuscript. C.S designed and performed most of the experiments. P.X. and X.L performed the chromatographic fingerprint analysis. W.J and D.X. assisted with data analysis and critically read the manuscript.
The authors gratefully acknowledge the financial support received from Research projects of traditional Chinese medicine of Hunan Province (202080, S.L) and Health Committee of Hunan Province (20200901, S.L.).
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