Characterization of the Components and Pharmacological Effects of Mountain-Cultivated Ginseng and Garden Ginseng Based on the Integrative Pharmacology Strategy

Panax ginseng C. A. Mey (PGCAM) is a herbaceous perennial belonging to the Araliaceae family, mainly including Mountain-Cultivated Ginseng (MCG) and Garden Ginseng (GG) on the market. We aimed to establish a rapid, accurate and effective method to distinguish 15-year-old MCG and GG using ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry (UPLC-QTOF-MS/MS), and also explored the pharmacological mechanisms of the main components using the Integrative Pharmacology-based Network Computational Research Platform of Traditional Chinese Medicine (TCMIP V2.0; http://www.tcmip.cn/). Altogether, 23 potential quality markers were characterized to distinguish 15-year-old MCG and GG, including ginsenosides Ra2, Rg1, and Ra1, and malonyl-ginsenoside Ra3, etc. The contents of 19 constituents (mainly protopanaxadiol-type) were higher in MCG compared with that in GG, and four constituents (mainly carbohydrate compounds) were higher in GG. The 105 putative targets corresponding to 23 potential quality markers were mainly involved in 30 pathways, which could be divided into 10 models, such as immune regulation, systems (metabolic, nervous, cardiovascular, reproductive), blood-pressure regulation, as well as antitumor, antiaging, antibacterial and anti-inflammatory effects. Furthermore, the potential quality markers of MCG and GG could inhibit the proliferation of breast cancer by regulating the mRNA expression of PSA, S6K, MDM2, and P53 genes by acting on AR, MTOR, PI3K and other targets. The Integrative Pharmacology Strategy may provide an efficient way to identify chemical constituents and explore the pharmacological actions of TCM formulations.

INTRODUCTION Panax ginseng C. A. Mey (PGCAM) is an herbaceous perennial belonging to the Araliaceae family. Its dried root and rhizome (Panax ginseng C. A. Mey. Radix et Rhizoma) have been used as a herbal medicine in China and some Asian countries for thousands of years (Mancuso and Santangelo, 2017), and have immunomodulation, antifatigue, antiaging, and anticancer effects (Shi et al., 2019). PGCAM was firstly recorded in Shennong's Classic of Materia Medica , during which all PGCAM grew wild. However, with the development of society, wild-growing PGCAM could not meet human requirements. To solve the problem, farmers began to study and explore suitable cultivation methods for PGCAM. There are four types of PGCAM according to different cultivation methods: wildgrowing ginseng, transplanted ginseng, mountain-cultivated ginseng (MCG) and garden ginseng (GG) (Yang et al., 2013). MCG and GG are recorded in the Pharmacopoeia of the People's Republic of China from 2005 edition. MCG and GG have become the main varieties on the market.
MCG refers to the seeds of PGCAM germinated and grown in tall mountains and dense forests for 10-20 years , so also called "Lin-Xia-Shan-Shen". If MCG grows long enough, its quality and efficacy become almost the same as that of wild PGCAM. GG refers to PGCAM that planted in a garden and harvested after 4-6 years. In general, the quality of MCG is higher than that of GG, which has a stronger pharmacological effect (Kim et al., 2020). The price of PGCAM is directly proportional to the grown period: the longer the period, the higher the price. Therefore, adulteration or falsification of PGCAM has always been a serious problem in the commercial market.
To solve this problem, researchers have proposed different identification strategies for PGCAM. With the continuous development of new technologies, ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry (UPLC-QTOF-MS/MS) has been widely used in the analysis of chemical components of PGCAM , multiple compounds have been identified, including ginsenosides, polysaccharides, fatty acids, volatile oils and amino acids (Liang et al., 2016). Ginsenosides, as the main components of PGCAM, show important pharmacological activities during the treatment of cardiovascular diseases (Kim, 2018). In addition, Prof. Xu and his colleague selected 12 chemical components to distinguish GG 4-7 years and MCG 15 years : ginsenoside Ra3/isomer, gypenoside XVII, quinquenoside R1, ginsenoside Ra7, notoginsenoside Fe, ginsenoside Ra2, ginsenoside Rs6/Rs7, malonyl ginsenoside Rc, malonyl ginsenoside Rb1, malonyl ginsenoside Rb2, palmitoleic acid, and ethyl linoleate . However, the characteristic components and pharmacological effects of MCG and GG grown for 15 years have not been reported. Therefore, we aimed to employ the Integrative Pharmacology Strategy to characterize the differential component between the two types of Ginseng and explore their pharmacological effects in vitro. Integrative Pharmacology Strategy (IPS) pays attention to the interactions between Chinese prescriptions and the organism from multiple levels and multiple aspects, its research content mainly includes component analysis, network pharmacology analysis and pharmacological experiment verification (Xu and Yang, 2014). The IPS systematically  analyzes the interaction between TCM formulations and the  organism from multiple levels and multiple aspects to form a  new mode of research of TCM. To practice this strategy better, we  established Integrative Pharmacology-based Network Computational Research Platform of Traditional Chinese Medicine (TCMIP V2.0; www.tcmip.cn/), which comprising five databases and seven functional modules . Therefore, we aimed to identify potential quality markers to distinguish 15-year-old MCG and GG using the UPLC-QTOF-MS/MS. Then, we employed the TCMIP V2.0 to carry out the network pharmacology analysis of the differential components contained in MCG and GG. Finally, we verified the results of network pharmacology in vitro. Our research will be a good application of Integrative Pharmacology Strategy.

Chemicals and Reagents
Twelve batches of MCG and GG were collected from the cultivation areas in Huanren County (41.26°N, 125.36°E; Benxi City, Liaoning Province, China) in September 2017, and the voucher specimens were deposited in our lab. Sixty-six standards of PGCAM were purchased from Shanghai Standard Biotech (Shanghai, China) or isolated from the roots of PGCAM and Panax notoginseng C. A. Mey (Yang et al., 2016b). (Supplementary Table S1). HPLC-grade acetonitrile (CH 3 CN) and methanol were purchased from Thermo Fisher Scientific (Fair Lawn, NJ, United States). Formic acid (FA) was obtained from Sigma-Aldrich (Saint Louis, MO, United States). Ultra-pure water was prepared in-house using the Milli-Q ™ system (Millipore, Bedford. MA, United States).

Sample Preparation
First, 50 mg of MCG powder and GG powder were soaked with 3 ml of 70% methanol (v/v), respectively, followed by ultrasound extraction for 60 min at 25°C. Then, the solution was centrifugated at 14,000 rpm for 10 min at room temperature after compensating with 70% methanol for the weight lost. The supernatant was transferred to a 5 ml volumetric flask and diluted to the scale mark. Finally, 1 mlwell-mixed liquid was centrifuged for 10 min, and supernatant were transferred to autosampler vials for analyses. Herbal samples were injected randomly. An equal volume of all test solutions was mixed to prepare the quality control (QC) sample, which was used to monitor the stability of the analytical system. 1.7 µm) hyphenated with a VanGuard ™ pre-column (2.1 × 50 mm, 1.7 µm; Waters) maintained at 35°C. The mobile phase consisted of 0.1% FA in CH 3 CN (A) and 0.1% FA in H 2 O (B). The optimized gradient program was: 0-2 min, 15-20% A; 2-7 min, 20-30% A; 7-17 min, 30-33% A; 17-20 min, 33-60% A; 20-22 min, 60-98% A; 22-24 min, 98-98% A. The flow rate was 0.3 ml/min and the injection volume was 3 μL. A 3-min re-equilibration time was set between successive injections. A "purge-wash-purge" cycle was set on the autosampler, with 10% CH 3 CN-H 2 O (v/v) as the purge solvent and 50% CH 3 CN-H 2 O as the wash solvent, to minimize the carry-over between injections.
The MS experiment performed on a Vion IMS-QTOF mass spectrometer in the negative electrospray ionization mode (Waters, Corp., Milford, United States). The LockSpray ™ ion source was equipped under the following parameters: capillary voltage, 1.0 kV; cone voltage, 20 V; source offset, 80 V; source temperature, 120°C; desolvation gas temperature, 500°C; desolvation gas flow (N 2 ), 800 L/h; cone gas flow (N 2 ), 50 L/h. Default parameters were defined for the traveling-wave IMS separation. HDMSE data covered a m/z of 300-1,500 at 0.3 s per scan. The low collision energy was set at 6 eV and the highenergy ramp was 40-80 eV. Data acquisition was controlled by the UNIFI 1.9.3.0 software (Waters, Corp., Milford, United States). The accuracy error threshold was fixed at 10 ppm.

Date Processing
Raw HDMS E data were corrected with reference to m/z 554.2620 in the 12 batches of samples and QC by UNIFI 1.9.3.0. Then, preliminary data were processed automatically by Progenesis QI 2.1 software (Waters, Corp., Milford, CT, United States) [M-H] − and [M + FA-H] − were the main adduct ions in the negative mode. Efficient menu-guided processing, peak alignment and peak selection could generate a data matrix, including retention time (t R ), m/z, normalized peak area, and collisioncross-secions (CCS). All the ion signals detected in each sample were normalized to the obtained value of the total ion count. The data matrix was filtered based on "80% rule" and "30% variation" .

The Untargeted Metabolomics Analysis Based on Multivariate Statistical Analysis
Processed data were subjected to principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA) using SIMCA-P 14.1 (Umetrics, Umea, Sweden). Potential quality markers were filtered out according to the variable importance for projection (VIP) values (VIP>1.5) and Student's t-test (p < 0.05), which could be used distinguish between MCG and GG.

Prediction of Putative Targets of Potential Quality Markers
The mol. formats of potential quality markers were uploaded to TCMIP to predict the putative targets using the TCM target prediction and function analysis module (TTFM) of TCMIP according to the similarity of chemical structures to known drugs on the market. The Tanimoto Score was set at 0.7 (moderate similarity) to select constitute-putative target pairs.

Pathway Enrichment Analysis
To further explore the biological functions of the putative targets, the pathway-enrichment analyses was undertaken using the database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8 (https://david.ncifcrf.gov). Pathways with p < 0.05 were selected for further analyses.
Cell Culture MCF-7 cell line was kindly provided by Prof. Xiujie Wang (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences). The MCF-7 cell line was cultured in DMEM high glucose medium (Thermo, Fair Lawn, NJ, United States) with 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, United States)and 10% fetal bovine serum (FBS; Sijiqing, Huzhou, Zhejiang, China) at 37°C in humid atmosphere with 5% CO 2 .

Cell Proliferation Analysis
The extraction of MCG and GG was prepared according to Sample Preparation. Then, the methanol was volatilized and the residue was redissolved with sterile water. After filtered by 0.22 μm filter film, the herbal samples were stored in a refrigerator at 4°C. MCF-7 cell line was seeded in 96-well plates (5×10 3 cells/ well) incubating at 37°C for 24 h. Various concentrations of MCG or GG were added to the wells and then incubated for 48 h. Cell proliferation was evaluated using a Cell Counting Kit-8 (CCK8) assay. The cells treated with various conditions were incubated with 10 μL CCK-8 solution (Dojindo, Kumamoto, Japan) for 90 min. Next the absorbance was measured by a Microplate Reader under 450 nm (Thermo, Fair Lawn, NJ, United States). All experiments were performed in triplicate and repeated four times. Data were presented as mean ± standard deviation (SD). A One-way ANOVA determined whether the results had statistical significance.

Differential Components Between MCG and GG Based on Untargeted Metabolomics
Multi-batch HDMS E data were processed by Progenesis QI, and generated a list comprising 1,023 ions (Supplementary Table  S2). These ions were filtered based on "80% rule" and "30% variation" (Supplementary Table S3). Then, the remained 954 ions were subjected to PCA and OPLS-DA. MCG and GG groups were separated well in PCA score Plots (Figure1A), indicating the great difference in chemical profiles. To obtain better discrimination, they were subjected to OPLS-DA with R 2 X at 0.865 and Q 2 at 0.897, especially in the component P1+3 direction ( Figure 1B). The validity and predictability of OPLS-DA model was evaluated by 200-time permutation test ( Figure 1C). The regression lines of R2 and Q2 decreased with decreased in the correlation coefficients between permuted and original response variables. The result indicated that the predictive OPLS-DA models did not overfit. S-plots were obtained to show responsibility of each ion for these variations more showed intuitively the contribution of differential ions on distinguishing between MCG and GG. Most of the ions clustered around the original point ( Figure 1D), and only a few of them were scattered in the margin region. Just these few ions that representing chemical constituents contributed to the separation observed in the score plots. The ions that far away from the original point, always have higher value of VIP (Supplementary Figure S1). Therefore, 42 ions with VIP >1.5 and p < 0.05 (in the Student's t-test) were selected as potential quality markers (Supplementary

Identification of the Potential Quality Markers
Sixty-six standard compounds and our house library were helped to identify the 23 potential quality markers. The base peak intensity chromatograms of MCG and GG corresponding to negative ion mode were shown in       Table S1). Taking ginsenoside Ra2 as an example, the fragmentation pattern of panaxadiol saponins was analyzed.
Taking ginsenoside Rg1 as an example, the fragmentation pattern of panaxatriol saponins was analyzed.   Table S1).

Functional Analysis of Potential Quality Markers
A total of 105 putative targets were predicated based on 23 primarily identified compounds (Tanimoto score ≥0.7) using TTFM of TCMIP v2.0 (Supplementary Table S5). Which were mainly involved in 30 pathways after functional analysis DAVID 6.8 (Supplementary Table S6). The network of interactions between 23 components, the corresponding 105 putative targets and 30 pathways was visualized using Cytoscape v3.7.1 (Boston, MA, United States) as show in Figure 5. The 30 pathways were related to 10 function modules, such as metabolic system, nervous system, cardiovascular system, immune-regulation system and reproductive system. Among them, there were 12 immune pathways (fc epsilon RI signaling pathway; leukocyte transendothelial migration; non-small cell lung cancer; pathways in cancer; acute myeloid leukemia; toll-like receptor signaling pathway; small cell lung cancer; apoptosis; prostate cancer; natural killer cell mediated cytotoxicity; glioma; mammalian target of rapamycin signaling pathway (mTOR) signaling pathway), nine metabolic pathways (starch and sucrose metabolism; oxidative phosphorylation; type II diabetes mellitus (DM); insulin signaling pathway; galactose metabolism; tricarboxylic acid cycle (TCA cycle); glycolysis/  gluconeogenesis; fructose and mannose metabolism; amino sugar and nucleotide sugar metabolism), four nervous system pathways (Alzheimer's disease; Parkinson's disease; Huntington's disease; erbB signaling pathway), there cardiovascular system pathways (cardiac muscle contraction; contraction of vascular smooth muscle; aldosterone-regulated sodium reabsorption) and two reproductive-system pathways (progesterone-mediated oocyte maturation; Oocyte meiosis).
Inhibitory Effect of MCG and GG on MCF-7 Cell Line by Regulating the PSA, MDM2, P53, and S6K genes The enrichment analysis above showed that 23 components played more importment roles in tumor pathway, which indicated that the anticancer effect of MCG was stronger than GG. Therefore, we selected MCF-7 cell line to verify the results. The CCK-8 assay showed that MCG or GG did not significantly inhibit the proliferation of MCF-7 cell line when the concentration was lower than 4 mg/ml. When the concentration of MCG or GG was 8 mg/ml, the survival rate of MCG group was only 11.8%, and GG group was 21.99%, which indicated that the concentration of 4-8 mg/ml was the range of MCG and GG to inhibit the proliferation of MCF-7 cell line (Supplementary Figure S2). Thus, the concentrations of 4, 5, 6, 7, and 8 mg/ml were set to explore the inhibitory effect of MCG and GG on MCF-7 cell line. When the extract concentration was 4 mg/ml, the survival rate of MCF-7 cell line was significantly inhibited by MCG compared with GG ( Figure 6A).
Therefore, MG and GG at 4 mg/ml were selected to subject to RT-PCR detection. The mRNA expression of PSA, MDM2 and S6K genes in the group of MCG was significantly down-regulated compared with that of control group, and the degree of down regulation was stronger than that of GG. Conversely, the mRNA expression of P53 gene was significantly up-regulated, and the degree stronger than that of GG ( Figures 6B-E).

DISCUSSION
PGCAM as a herbal nutritional supplement is well known for its extensive pharmacological effects (Nguyen and Nguyen, 2019). MCG and GG are the main PGCAM varieties on the market to meet the huge demand of people (Yang et al., 2013) (Shen et al., 2019). At present, ginsenoside is the main marker for identification of different species of MCG and GG. For example, ginsenosides can be used to distinguish different planting methods and different years of ginseng (Cui et al., 2013;Ruan et al., 2018). As natural plants, the environment and duration of growth affect the quality and price of PGCAM directly. Zhu et al., 2018). Many studies have been undertaken to characterize the chemical profiles of PGCAM cultivated in different environments for various durations, which could help distinguish diverse samples (Chang et al., 2016;Chang et al., 2017). Hence, we aimed to employ integrative pharmacology strategy to characterize the differential components between the two types of Ginseng and explore their pharmacological effects by network pharmacology and verified in vitro.
Altogether, 23 chemical components were identified to distinguish the two sets of samples, and nine of them were identified according to use of reference compounds. The characteristic fragments of PPD-saponins were m/z 459.38 and m/z 375.29. These data could be used to identify unknown marker compounds belonging to this type, including M14, M16, M18, M26, M29, M30, M36, M39, and M41. Ginsenosides were the main characteristic components of the MCG group. Carbohydrates were the main characteristic components of the GG group. The peak area of PPD-type saponins in the MCG group was higher than that of GG group, and the peak area of carbohydrate compounds was higher in the GG group. The results showed that ginsenoside Ra2 and sucrose could not only distinguish 15 years old MCG and GG, but also 10-20 years old MCG and 4-6 years old GG . Ginsenoside Rg1 can not only distinguish the 15-year-old forest ginseng and garden ginseng, but also distinguish the ginseng of different ages, different planting sites and different slope directions (Zhu et al., 2021). In conclusion, some of the quality markers in the same region can distinguish ginseng under different conditions. It shows that our research results have certain universality.
To explore further the pharmacological activities of these marker components, components-targets-pathways network was constructed. We found that the marker components could regulate immune, metabolic, nervous-system, cardiovascular-system and reproductive-system pathways. Among them, immunomodulatory and metabolic regulatory pathways were the main pathways regulated by marker components during the treatment of cancer and diabetes mellitus.
The pharmacological effects of marker components were investigated further. Ginsenoside Rg1 has been found to: 1) protect the heart from cardiovascular diseases ; 2) impact the neuroendocrine system for treatment of depression (Mou et al., 2017); 3) inhibit inflammation and apoptosis (Guo et al., 2019); 4) possess neuroprotective properties (Li G. et al., 2019); 5) enhance gene expression and oxidative muscle metabolism in muscles (Jeong et al., 2019); 6) protect against diabetic nephropathy (DN) by reducing oxidative stress (Du et al., 2018). Hence, Rg1 can regulate the cardiovascular system, nervous system and immune system. Sucrose can reduce procedural pain from single events, including heel lancing, venipuncture and intramuscular injection, which shows that sucrose can regulate the nervous system without side effects. (Stevens et al., 2016). Ginsenoside Ra1 is the main active component of ginseng used for immune regulation . Hence, ginsenosides Ra1 can affect the cardiovascular system and regulate the immune system. Notoginsenoside K has been shown to exhibit immunologicadjuvant activities on the humoral immune responses of ICR mice against ovalbumin (Sun et al., 2005;Qin et al., 2006). Ginsenosides Ra1, notoginsenoside K, and ginsenoside Rg1 can affect the cardiovascular system, immune regulation, nervous system, and have anti-inflammatory and antioxidant effects. Sucrose has a protective effect upon HepG2 cells and fibroblasts (Sim et al., 2020), which suggests that sucrose has an antioxidant effect. Maltose can enhance the anti-melanogenic activity (Bin et al., 2017). The results showed that the pharmacological activities of potential quality markers were consistent with those reported in the literature. Seven malonylginsenosides (malonyl-ginsenoside Ra1, malonylginsenoside Ra2, malonyl-ginsenoside Ra3, malonylfloralginsenoside Rd, malonyl-floralginsenoside Rd6, malonylfloralginsenoside Re1 and malonyl-floralginsenoside Rd5) were identified as the PPD-type markers of MCG and GG. However, reports on the pharmacologic effects and mechanism of action of malonyl saponins are lacking, which needed further study. Network pharmacology pathway enrichment showed that the cancer pathways enriched by quality markers were more abundant. Therefore, we used MCF-7 cell line to evaluate the anticancer effect of MCG and GG. The results showed that the anticancer effect of MCG was better than GG, which was consistent with the published data (Kim et al., 2020). The main genes involve in anticancer effect of MCG and GG are PIK3CA, PIK3CG, MTOR and AR, which are also the key genes of PI3K/Akt/mTOR pathway. Among them, the quality markers acting on PIK3CA, PIK3CG and AR were malonyl -ginsenoside Ra3, malonyl-floralginsenoside Rd, malonyl -ginsenoside Ra1, malonyl-floralginsenoside Rd6, malonyl -ginsenoside Ra2, malonyl-floralginsenoside Re1, malonyl-floralginsenoside Rd5, notoginsenoside Rt, notoginsenoside S, notoginsenoside T, notoginsenoside K, ginsenoside Ra1, ginsenoside Ra2, ginsenoside Ra3, Ginsenoside Rg1. The potential quality markers acting on MTOR were malonyl -ginsenoside Ra3, malonyl-floralginsenoside Rd, malonyl -ginsenoside Ra1, malonyl-floralginsenoside Rd6, malonyl -ginsenoside Ra2, malonyl-floralginsenoside Re1, malonyl-floralginsenoside Rd5, which were common quality markers of PIK3CA, PIK3CG, MTOR. Except Notoginsenoside Rt, the content of the remaining 14 quality markers in MCG was higher than GG. In conclusion, the 14 quality markers of MCG play a better role in anticancer by acting on PI3K/Akt/mTOR pathway while the data of mRNA expression of PIK3CA, PIK3CG, MTOR, AR in the MCF-7 cell samples treated by MCG and GG showed that there was no significant difference between ginseng treatment group and control group. However, we found that downstream genes of the PI3K pathway play an important role in cancer therapy through literature research. In addition, MDM2 has been shown to be abnormally upregulated due to gene amplification, increased transcription, and enhanced translation, leading to enhanced degradation and reduction of P53 activity in some human tumors (Konopleva et al., 2020)and therefore, many drugs/compounds have been developed for reactivation of P53 gene by inhibiting MDM2 interaction with P53 in order to treat cancer (Gupta et al., 2019). Moreover, MTOR pathway plays an important role in the treatment of human cancer. mTORC1 mediates its function via its downstream targets 40 S ribosomal S6 kinases (S6K) (Sridharan and Basu, 2020). Prostate specific antigen (PSA) is a serine protease produced by prostate epithelial cells and prostate cancer (PCA), which can be regulated by AR. The increase of PSA level can be used as a diagnostic marker of tumor or cancer (Balk et al., 2003). The RT-PCR data showed that mRNA expression of MDM2, S6K, PSA were significantly decreased, and the mRNA expression of P53 gene was increased in MCG compared with GG. In summary, the results show that the quality markers of MCG can reduce the survival rate of breast cancer cells better by acting on PIK3CG, PIK3CA, mTOR, AR genes and co-regulating the effector genes MDM2, P53, PSA, S6K ( Figure 6F).

CONCLUSION
We characterized, for the first time, 23 chemical components to distinguish 15-year-old MCG and GG, which corresponding to 105 putative targets and 30 pathways. In addition, the 23 components inhibit the proliferation of breast cancer (MCF-7 cells) by regulating the mRNA expression of PSA, S6K, MDM2 and P53, in which MG exhibited stronger effects than that of GG. Integrative Pharmacology Strategy undoubtedly provide an efficient way to identify chemical constituents and explore the pharmacological actions of TCM formulations.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
HX and LH designed the study. SL and PW drafted the manuscript. SL and WY analyzed the chemical constituents in PGCAM. SL analyzed of network pharmacology. SL, CZ, and LZ carried out anticancer pharmacodynamics experiment and RT-PCR detection. JZ and YQ established the UNIFI database of PGCAM. The other authors participated in the design of the study and performed the statistical analysis All authors read and approved the final version of the manuscript. Research Institutes (ZXKT17058). These funding agencies had no role in the study design, collection, analyses, or interpretation of data, writing of the report, or the decision to submit the manuscript for publication.