*4.7. Untargeted Metabolomics by Liquid Chromatography Coupled Mass Spectrometry (MS/MS)* See details in Methods S1.

#### *4.8. Lactate Measurement*

For lactate measurement in serum, the whole blood of treated mice was firstly collected and centrifuged at 4000 rpm for 10 min to obtain the serum. For the analysis of lactate in cell cultures, cells were seeded at approximately 3 <sup>×</sup> <sup>10</sup><sup>5</sup> in 6-well plates and treated with different concentrations of ASP and/or AMP for 3 days. After centrifugation, the culture medium and cells were collected. The lactate content was estimated using the L-lactate assay kit (Jiancheng Bioengineering, Nanjing, China) according to the manufacturer's protocol. Lactate dehydrogenase catalyzed the conversion of lactate to pyruvic acid, and the absorbance at 530 nm was determined spectrophotometrically after the addition of a color developing agent, which showed a linear relationship with lactate content. Three replicates were performed, and the experiments were repeated three times for accuracy.

#### *4.9. Western Blot*

Total cell protein was collected (KGP2100, KeyGEN BioTECH, Beijing, China), and its quantification was performed using the BCA (cat. no. KGP902, KeyGEN BioTECH, Beijing, China) method. Proteins were separated by SDS-PAGE and transferred through PVDF membranes. After blocking the membrane with 5% BSA for 2 h at room temperature, membranes were incubated overnight at 4 ◦C with one of the following primary antibodies: rabbit monoclonal anti-STAT3 (ab68153, Abcam, Cambridge, UK), rabbit monoclonal antiphospho STAT3 (ab32143, Abcam, Cambridge, UK), rabbit monoclonal anti-Janus kinase 2 (JAK2, ab108596, Abcam, Cambridge, UK), rabbit monoclonal anti-phospho JAK2 (ab32101, Abcam, Cambridge, UK), rabbit monoclonal anti-proliferating cell nuclear antigen (PCNA, ab92552, Abcam, Cambridge, UK), rabbit monoclonal anti-MYC (ab32072, Abcam), mouse monoclonal anti-P53 (ab90363, Abcam, Cambridge, UK), rabbit monoclonal anti-β-actin (Bsm-33036M, Biosynthesis, Beijing, China), rabbit monoclonal anti-HK2 (ab209847, Abcam, Cambridge, UK), and rabbit monoclonal anti-glucose transporter 1 (GLUT1, ab115730, Abcam, Cambridge, UK). All antibodies were diluted at 1:1000 in TBST (T1086, Solarbio, Beijing, China). After washing with PBS, membranes were incubated with secondary antibodies, including goat anti-mouse IgG antibody (bs-0296G, Bioss, Beijing, China) and

goat anti-rabbit IgG antibody (bs-0295G; Bioss, Beijing, China), which were diluted to 1:4000 by TBST, for 1 h at room temperature. After washing off the excess secondary antibody, protein intensity was determined with Clarity Western ECL Substrate (Bio-Rad Laboratories Co. Ltd., Hercules, CA, USA) and measured by Image Lab software (5.2.1 Version, Bio-Rad Laboratories Co. Ltd., Hercules, CA, USA). Proteins were quantified by densitometric analysis of ImageJ (version 1.8.0.172, Bethesda, MA, USA).

#### *4.10. Histology and Immunohistochemistry (IHC) Analysis*

Murine liver tissues were collected, fixed in 4% paraformaldehyde for 48 h, dehydrated, and embedded in paraffin. The paraffin tissue blocks were cut into 4 µm-thick sections, deparaffinized with xylene and passed through graded alcohol (10009218, ChengFeng Co., Ltd., Hangzhou, China). To detect the expression of HK2 and Ki-67 in liver tissue, sections were incubated with anti-HK2 antibody (1:200, ab209847, Abcam, Cambridge, UK) or anti-Ki67 antibody (1:200, bs-2130R, Bioss, Beijing, China). Sections were incubated overnight at 4 ◦C and rinsed 3 times with 1 × PBS for 5 min each time. Reagents were added according to the instructions of the secondary antibody kit (sp-9000; ZSGB-Bio, Beijing, China), and finally DAB (ZLI-9018; ZSGB-Bio, Beijing, China) were added for a 2 min reaction. Four views were randomly collected from each section, and images were acquired using an optical microscope (DM3000, Leica Co., Ltd., Weztlar, Germany).

#### *4.11. Real-Time Quantitative PCR Analysis*

Total cellular RNA was extracted by using Trizol Reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Reverse transcription was performed using Prime-Script (Takara Bio, Inc., Nojihigashi Kusatsu, Japan), first removing gDNA by using the following conditions: 42 ◦C for 2 min. The conditions used for reverse transcription were as follows: 37 ◦C for 15 min and 85 ◦C for 5 s. The cDNA was subjected to PCR by adding SYBR Green (Takara Bio, Inc., Nojihigashi Kusatsu, Japan), and the sequences of the primers used are shown in Table 1. Real-time quantitative PCR detection was performed by using a CFX96 Deep well (Bio-Rad Laboratories, Inc., Hercules, CA, USA) under the following cycling conditions: 95 ◦C for 30 s, 95 ◦C for 5 s, and 60 ◦C for 30 s, with 40 cycles. Experiments normalized the mRNA expression of each gene with β-actin as an internal control. All samples were repeated three times. Finally, the relative gene expression results were calculated using the 2−∆∆Ct method.


**Table 1.** Primers which have been used for real time PCR.


#### **Table 1.** *Cont.*

*4.12. Network Pharmacology and Bioinformatics*

See details in Methods S2.

#### *4.13. Statistical Analysis*

Statistical analysis was performed using SPSS version 22.0 (IBM, Corp., Armonk, NY, USA). All the data are expressed as mean ± standard deviation. One-way ANOVA or unpaired two-tail Student's t-test was used to determine differences among groups. A statistically significant difference was considered significant at *p* = 0.05, \* *p* < 0.05, \*\* *p* < 0.01, and \*\*\* *p* < 0.001.

#### **5. Conclusions**

The present investigations demonstrated that ASP-AMP acted as a promoting factor for liver regeneration. ASP-AMP intervention in hepatocytes enhanced proliferation by accelerating HK2-associated glycolysis. Moreover, ASP-AMP-induced glycolysis involved liver regeneration by JAK2/STAT3/HK2 signaling. These findings demonstrate that ASP and AMP, as natural agents, are feasible and experimentally recommended in the treatment of liver regeneration in patients who undergo hepatectomy. To impel the use of ASP-AMP in clinic therapy, the pharmacokinetics and toxicity should be further investigated. In addition, several missing links remain to be elucidated in future studies, including the molecular mechanism responsible for the ASP-AMP-mediated upregulation of JAK2 and the STAT3-induced HK2 activation.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27227890/s1, Method S1: Untargeted metabolomics by liquid chromatography coupled mass spectrometry (MS/MS); Method S2: Network pharmacology and bioinformatics; Figure S1: *Angelica sinensis* polysaccharide (ASP) and *Astragalus membranaceus* polysaccharide (AMP) promoted the proliferation of BRL-3A; Figure S2: Combined use of both ASP and AMP enhanced the proliferation of BRL-3A; Figure S3: Volcanic map of differential metabolites; Figure S4: Potential targets analysis of drugs (ASP and AMP) and liver regeneration.

**Author Contributions:** Conceptualization, Y.-H.H., T.P. and Q.-B.Y.; data curation, X.-D.W.; formal analysis, X.-D.W., Y.-L.Z. and L.Y.; funding acquisition, X.-D.W. and Q.-B.Y.; investigation, Z.Y. and Q.-B.Y.; methodology, X.-D.W., Y.-L.Z., L.Y. and Z.Y.; resources, Y.-L.Z.; software, G.-C.F.; validation, L.Y. and Z.Y.; writing—original draft, X.-D.W. and Y.-L.Z.; writing—review and editing, T.P. and Q.-B.Y. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China, grant number 81973742; Experimental Formulary Sichuan Youth Science and technology Innovation research team, grant number 2020JDTD0022; XingLin Scholars Program of Chengdu University of TCM, grant number QJJJ2022002, YYZX2020036 and Sichuan Provincial Science and Technology Department, grant number 2021YJ0198.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki, and approved by the Animal Research Ethics Committee of Chengdu University of Traditional Chinese Medicine (protocol code 2022-0342, 14 March 2022) for studies involving animals.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available upon request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.

**Sample Availability:** Samples of ASP and AMP are available from the corresponding author.

#### **References**

