Abstract Polygonatum sibiricum Red. has been used as a medicinal herb and nutritional food in traditional Chinese medicine for a long time. It must be processed prior to clinical use for safe and effective applications. However, the present studies mainly focused on crude Polygonatum sibiricum (PS). This study aimed to investigate the chemical properties, blood-enriching effects and mechanism of polysaccharide from the steam-processed Polygonatum sibiricum (SPS), which is a common form of PS in clinical applications. Instrumentation analyses and chemistry analyses revealed the structure of SPS polysaccharide (SPSP). A mice model of blood deficiency syndrome (BDS) was induced by acetylphenylhydrazine (APH) and cyclophosphamide (CTX). Blood routine test, spleen histopathological changes, serum cytokines, etc. were measured. The spleen transcriptome changes of BDS mice were detected by RNA sequencing (RNA-seq). The results showed that SPSP consists predominantly of Gal and GalA together with fewer amounts of Man, Glc, Ara, Rha and GlcN. It could significantly increase peripheral blood cells, restore the splenic trabecular structure, and reverse hematopoietic cytokines to normal levels. RNA-seq analysis showed that 122 differentially expressed genes (DEGs) were obtained after SPSP treatment. GO and KEGG analysis revealed that SPSP-regulated DEGs were mainly involved in hematopoiesis, immune regulation signaling pathways. The reliability of transcriptome profiling was validated by quantitative real-time PCR and Western blot, and the results indicated that the potential molecular mechanisms of the blood-enriching effects of SPSP might be associated with the regulating of JAK1-STAT1 pathway, and elevated the hematopoietic cytokines (EPO, G-CSF, TNF-α and IL-6). This work provides important information on the potential mechanisms of SPSP against BDS. Keywords: steam-processed Polygonatum sibiricum polysaccharide, blood deficiency syndrome, RNA-seq, JAK1-STAT1, hematopoietic cytokines Introduction In Traditional Chinese Medicine (TCM), blood deficiency is considered to be related to deficiency in the stomach and spleen, and insufficient hematogenesis, which resembles to the symptoms of anemia in modern medicine ([37]1). TCM considers Qi and blood as the primary factors of blood deficiency, and blood deficiency syndrome (BDS) is a pathological state caused by weakness of spleen, excessive blood loss, andaplastic anemia, and other blood diseases ([38]2). In clinal, patients such as postoperative and postpartum women with chronic bleeding, and women with excessive menstruation usually have symptoms similar to BDS ([39]2). It is usually diagnosed by lower amounts of hemoglobin, blood cells and so on ([40]3). Hematopoietic cytokines, such as thrombopoietin (TPO), erythropoietin (EPO), interleukin-6 (IL-6), and granulocyte colony stimulating factor (G-CSF), play vital roles in the progress of haemopoiesis ([41]4). Oral iron supplements are the most conventional and effective agents to cure BDS. However, its application is limited for the adverse effects, such as gastrointestinal discomfort and exacerbate inflammatory bowel disease in clinic ([42]5). In recent years, several Chinese medicinal herb recipes that can enrich and regulate blood, such as E’jiao, Panax notoginseng, Angelica sinensis, Siwu Decoction have been proved that they have promising effects on prevention and treatment of BDS as well as anemia ([43]6–[44]9). The dry rhizome of Polygonatum sibiricum Red. is called as Polygonatum sibiricum (PS). It possesses a strong effect on invigorating Qi, nourishing Yin, moistening the lung, and tonifying spleen and kidney. Besides, PS can be used to treat coughing, weakness, fatigue, indigestion, premature graying of hair, anti-aging, etc. ([45]10). Since its beneficial effects on prolonging one’s life, PS is considered as the “Top grade” herbs in Shennong Bencao Jing, one of the most respected Chinese classics about medicinal plants. Numbers of active compounds have been isolated from PS, mainly including polysaccharides, monosaccharides, saponins and many other bioactive substances ([46]11). Specifically, polysaccharides of PS exhibited many biological activities such as anti-tumor ([47]12), enhancing immunity ([48]13, [49]14), antioxidant ([50]13, [51]15), regulating blood glucose and lipids ([52]13, [53]16), lung protection activities ([54]17), etc. The mixture of fructose, glucose, and sucrose in PS was reported to have blood tonic activity ([55]18). For safe and effective applications, PS must be processed prior to clinical use, which usually processed by steam or rice wine to eliminate the side effects of numbing ([56]19). A study had reported that the types and molar ratios of monosaccharides were different between wine-processed PS polysaccharides (WPSP) and crude PS polysaccharides (CPSP), and the immunological activity in vitro and vivo of WPSP was significantly better than that of CPSP ([57]20). Clinical studies have demonstrated that steam-processed PS (SPS) could enhance the original function of “invigorating qi and blood, and tonifying the spleen, kidney and liver” ([58]21). An animal study revealed that SPS could significantly increase WBC, RBC, HGB, and PLT in peripheral blood of BDS mice, while there were not significantly improved in crude PS ([59]22). Nevertheless, little is known regarding the chemical properties of SPS polysaccharides (SPSP), and the potential molecular mechanism on BDS. Therefore, the chemical characteristics of SPSP and its mechanism on blood deficiency are very worthy of further research. In this study, instrumentation analyses and chemistry analyses were utilized together to identify the structure of SPSP. Cyclophosphamide (CTX) and acetyl phenylhydrazine (APH) were used to establish the haemolytic, aplastic anaemia and BDS model ([60]23, [61]24). The effects of SPSP on BDS were evaluated with a classical BDS model induced by CTX and APH in this study. Transcriptomics was used to explore the potential mechanism of SPSP on BDS mice. Materials and Methods Materials and Chemicals The steam-processed Polygonatum sibiricum was provided by Zhejiang Sanxitang Traditional Chinese Medicine Co., Ltd (No. Y2012301, Wuyi, China). Voucher specimens were deposited at Zhejiang Pharmaceutical College, Ningbo, China. CTX was obtained from Hengrui Medicine Co., Ltd. (Jiangsu, China). APH was purchased from Aladdin Biochemical Technology Co., Ltd (Shanghai, China). ELISA detection kits for G-CSF, tumor necrosis factor-α (TNF-α), IL-6, and EPO were purchased from Shanghai Jianglai Biological Technology Co., Ltd. (Shanghai, China). Extraction, Isolation, and Purification of SPSP 1.2 kg steam-processed Polygonatum sibiricum were soaked in deionized water at a ratio of 1:10 (w/v) for 12 h and refluxed twice at 80°C, 1.5 h each. The extracts were combined together and concentrated to a suspension of a solid–liquid ratio of 1:1 by a rotary evaporator at 60°C. Anhydrous ethanol was slowly added to the suspension to make its final concentration be 80%. Then, the suspension was stored at 4°C overnight to obtain the precipitates. The precipitates were collected by centrifugation and heated on a 60°C water bath to remove the residual ethanol. Subsequently, the precipitates were dissolved in an appropriate amount of ultrapure water, and deproteinized using Sevag reagent (n-butanol:chloroform = 1:4, v/v). The deproteinized solution was dialyzed with running distilled water (cut-off Mw: 8000 Da) for 72 h. Following that, the deproteinized samples were observed on a UV spectrophotometer, which revealed no absorbance peaks at 280 nm. Finally, a SPSP sample was obtained through vacuum freeze drying for subsequent experiments. Partial Structural Characterization of SPSP Molecular Weights (Mw) The Mw of SPSP was measured using high performance gel permeation chromatography (HPGPC) system following a previously reported method ([62]25). SPSP was dissolved in 0.02 M phosphate buffer (pH 6.8) at a concentration of 5 mg/mL. Dextran standards with different Mw (1152, 11600, 23800, 48600, 80900, 148000, 273000, and 409800 Da) were used to obtain the standard calibration curve. Both the sample and standard solutions were centrifugated at 13000 r·•min^-1 for 10 min, and 20 μL of supernatant were injected into a HPGPC instrument (Column: Shodex SB-804, 300×8 mm), respectively. The column temperature was kept constant at 25°C, and the mobile phase was phosphate buffer at a flow rate of 0.5 mL•min^-1. Monosaccharide Composition Analysis 3 M trifluoroacetic acid (TFA) solution was used to dissolve SPSP (10 mg), and then the solute (1 mg/mL) was hydrolyzed at 120°C for 3 h. A rotary evaporator was used to evaporate the hydrolysate before mixing with methanol, followed by another evaporation session to complete dryness with a termovap sample concentrator. Then the residue was dissolved in distilled water (5 mL), and diluted to 50 mL. The Ion chromatography (IC) measurements was performed with a Dionex ICS5000 system (ThermoFisher, USA) equipped with an anion exchange column (Dionex CarboPac PA20, 3 mm×150 mm). The column temperature was kept constant at 30°C. Sixteen standard monosaccharides, namely fucose (Fuc), rhamnose (Rha), arabinose (Ara), N-acetyl-D-glucosamine (GlcNAc), galactose (GalA), mannose (Man), glucose (Glc), xylose (Xyl), mannuronic acid (ManA), fructose (Fru), ribose (Rib), Galactosamine hydrochloride (GalN), Glucosamine hydrochloride (GlcN), guluronic acid (GulA), glucuronic acid (GlcA), and galacturonic acid (GalA) were used as the references.