Along the gut-bone marrow signaling pathway: use of longan polysaccharides to regenerate blood cells after chemotherapy-induced myelosuppression

Abstract

Although it has been established that polysaccharides have an effect on bone marrow haematopoiesis, it remains unclear how polysaccharides regulate bone marrow haematopoiesis during absorption and metabolism in vivo. In this study, the effect of a longan polysaccharide of large molecular weight (TLPL) on the gut microbiota of mice and its implications for the haematopoietic process in bone marrow was discussed. Here, the results show that after 21 days of TLPL consumption, the respective quantities of white blood cells, platelets, hemoglobin and bone marrow nucleated cells were determined to be 3.18 ± 1.71 (10⁹ L⁻¹), 1238.10 ± 164.41 (10⁹ L⁻¹), 135.10 ± 4.95 (g L⁻¹), and 1.70 × 10⁷, which reached 56.98%, 117.28%, and 47.74%, respectively, of the results for NC. TLPL both increased the thymus and spleen indexes by up to 2.08 ± 0.64 (mg g⁻¹) and 6.49 ± 2.45 (mg g⁻¹), respectively. Additionally, TLPL remodeled the gut microbiota with a significant increase in Lactobacillus in particular, and a significant increase in the level of the potential intestinal metabolite lactate was detected in the serum. Most importantly, a similarly significant up-regulation of the gene expression of the lactate receptor, Gpr81, in the myeloid cells was observed. These changes contributed to the activation of the secretion of various cytokines associated with haematopoiesis, with the levels of G-CSF, EPO, SCF and PF4 increased by 2.44 times, 1.14 times, 1.56 times and 1.13 times, respectively, compared to the MC group, which subsequently accelerated production of bone marrow cells and blood cells. The findings of this study reveal the unique mechanism of dried longan polysaccharides in ameliorating myelosuppression and provide a feasible strategy for the treatment of chemotherapy-induced myelosuppression with bioactive polysaccharides.

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1 Introduction

The incidence of malignant tumours continues to rise and has become a globally recognised widespread disease that poses a serious threat to human health.¹ Currently, chemotherapy remains the mainstay of treatment for advanced malignant tumours.² However, cyclophosphamide (CTX), a drug widely used in cancer treatment, affects the stability of bone marrow stromal cells and haematopoietic regulators, and disturbs the microenvironment of bone marrow stem cells.³ New approaches are urgently needed to ameliorate the immune system for the prevention of chemotherapy-induced bone marrow suppression.

It is well known that natural products contribute to the development of medicinal biology. Alternative therapeutic systems using natural products from medicinal and food sources and their isolated active ingredients have aroused the interest of Chinese researchers.⁴ Numerous studies at home and abroad have shown that polysaccharides from traditional Chinese medicine sources (Angelica sinensis, Radix Astragali, Polygonatum sibiricum, Ganoderma lucidum, etc.) are able to effectively alleviate all types of haematopoiesis induced by chemotherapy and to promote haematopoietic function restoration, and are not accompanied by any significant adverse effects.^5–8 However, the investigation of the mechanisms of these herbal polysaccharides is limited to anti-haematopoietic cell apoptotic signals, attenuation of oxidative damage and reduction of the body's inflammatory response, etc.,^6,9–11 without considering the complex metabolism of polysaccharides in the intestine, i.e., analysing whether they regulate the gut microbiota from the point of view of the function. Collectively, considering that polysaccharides are difficult to be absorbed directly through the intestine, this implies that the bioactive functions of polysaccharides involve the utilisation of gut microbiota. As such, this aspect undoubtedly provides new ideas for studying the pro-haematopoietic functions of plant polysaccharides.

Dried longan (Gui Yuan) is considered to have the effect of “benefiting the spleen and replenishing blood” in traditional Chinese medicine, and “replenishing blood” is interpreted as “haematopoiesis” in modern pharmacology.¹² Longan polysaccharides have been shown to confer a variety of health benefits, which has led to conjecture that they could potentially be active substances that help the body regulate haematopoiesis. Many researchers and scholars have demonstrated, at both the cellular and animal levels, that the immunoactive substances in dried longan are the large molecular weight polysaccharides.^13,14 These polysaccharides are able to protect the organ health of immunosuppressed mice and promote the recovery of peripheral blood volume. These beneficial results make it possible to explore the pro-haematopoietic effects of dried longan polysaccharides, as organismal immunosuppression occurs in the presence of myelosuppression.¹⁵ Moreover, longan polysaccharide is not digestible by humans, and after ingestion it enters the large intestine where it is degraded and fermented by the gut microbiota.¹⁶ Gut microbiota has been shown to be involved in the immunomodulatory activity of dried longan polysaccharides, whose ingestion leads to an increase in the levels of Lactobacillus, Pediococcus, and Bifidobacterium, as well as an increase in short-chain fatty acid levels in the colon.^17,18 The gut bacteria have been reported to play a key role in regulating haematopoiesis in organisms in recent years.^19,20 Microbial metabolites enter the blood circulation and contribute to the haematopoietic process through a variety of key signals.²¹ Some gut microbiota metabolites such as lactate, NOD1L, and butyric acid have been reported to have the ability to stimulate haematopoietic-associated cells, accelerating haematopoiesis and erythropoiesis.^22–24 Thus, the basis of these previous studies made it possible to explore whether dried longan polysaccharides would improve haematopoiesis in myelosuppressed mice by modulating gut microbiota.

Despite these previous works, the mechanisms by which dried longan polysaccharides affect the microbiota contributing to haematopoiesis in myelosuppressed mice remain poorly understood. Specifically, do longan polysaccharides have haematopoietic activity? By what mechanism does the microbiota signal to haematopoietic cells in the distal bone marrow? How are haematopoietic cells translated to influence haematopoietic activity? Current research is focused on determining the molecular mechanisms by which dried longan polysaccharides modulate the microbiota to promote normal haematopoiesis, a necessary step to further inform the development of functional foods for myeloprotection and haematopoiesis that have a clear material basis, a clear mechanism, and minimal adverse effects.

2 Materials and methods

2.1 Materials and chemicals

Longans (Dimocarpus longan Lour. cv. Chuliang) were purchased from a local orchard (Deqing, Guangdong province, China) in 2018. The fresh longan fruits were dried using a heat pump at 65 °C for 48 h.

The enzyme-linked immunosorbent assay (ELISA) test kits for granulocyte-colony stimulating factor (G-CSF), erythropoietin (EPO), stem cell factor (SCF) and platelet factor 4 (PF4) were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. Tumor necrosis factor-α (TNF-α) and interleukin-γ (IFN-γ) were purchased from NeoBioscience Technology Co., Ltd. The superoxidase dismutase (SOD) assay kit was from Suzhou Grise Biotechnology Co. Ferric reducing antioxidant power (FRAP) and malondialdehyde (MDA) assay kits were from Beijing Biyuntian Biotechnology Co., Ltd. Cyclophosphamide (CTX) was purchased from Sigma-Aldrich Medical Pharmaceutical Co., Ltd.

2.2 Extraction and isolation of TLPL

Dried longan pulps (50 g) were obtained after removing the seed and pericarp, and the pulps were extracted with 4.5 L of distilled water at 80 °C for 1.5 h. The pomace was removed using a gauze, and the extracting solution was concentrated to 1.5 L by a rotary evaporator at 60 °C. Then, three-phase partitioning for the extraction and separation of crude longan polysaccharides was performed as described by Yan et al. (2018) with some minor modifications.²⁵ Briefly, 20% (w/w) (NH₄)₂SO₄ was added to the resulting supernatant and stirred until completely dissolved. Subsequently, the mixture was mixed with t-butanol (1 : 1, v/v) and continuously stirred at 40 °C for 30 min. Extracts with molecular weights greater than 100 kDa were collected using ultrafiltration membranes (Sartorius Scientific Instruments (Beijing) Co., Ltd, Vivaflow 50R), lyophilized and named as a three-phase-partitioned longan polysaccharide of large molecular weight (TLPL).

2.3 Molecular weight, monosaccharide composition and structure analysis of TLPL

High-performance gel-permeation chromatography (HPGPC) was used to determine the molecular weight (MW) distribution of TLPL, and the seven dextran standards with different molecular weights ranging from 6200–488 000 kDa. At the same time, a UV-Vis spectrophotometer was used to scan the samples in the range of 200–500 nm for the detection of proteins and other impurities, and a Congo Red assay was used to detect the conformation of TLPL. The monosaccharide composition of TLPL was determined by ion chromatography (IC) as described by Hui et al. (2019).²⁶ TLPL powder was placed in the cuvette of the diamond single-reflection ATR accessory fitted to the infrared spectrometer to a thickness of about 0.2 cm. The FT-IR spectrum measurement range was 4000–600 cm⁻¹.

2.4 Animals and experimental design

Male Balb/c mice of 6–8 weeks and 18–22 g in weight that were of specific pathogen-free (SPF) grade were obtained from Hunan SJA Laboratory Animal Co., Ltd (Certificate No.: SCXK (Xiang) 2021-0002). All animal experiments were performed in accordance with the guidelines of the Experimental Animal Ethics Committee of South China Agricultural University (Animal experiment ethics SYXK [Yue] 2019-0136). After 1 week of acclimatization, the mice were randomized into four groups with equal numbers in each group as the negative control (NC) group, the model control (MC) group, the TLPL group and the recombinant human granulocyte colony-stimulating factor (rhG-CSF) group. rhG-CSF mainly acts on granulosa lineage haematopoietic progenitor cells in the bone marrow to promote their proliferation and differentiation, and to increase the function of granulosa lineage terminally differentiated cells.

The MC group, TLPL group and rhG-CSF group were intraperitoneally (IP) injected with cyclophosphamide (CTX) that was dissolved in normal saline (NS) at 100 mg kg⁻¹ for 3 consecutive days to establish the myelosuppression model while mice in control group were IP injected with NS for 3 days. Afterwards, 80 mg kg⁻¹ CTX was supplemented with 1 injection per week to prevent haematopoietic recovery. The mice in the NC and MC groups were treated with 10 mL kg⁻¹ double distilled (D.D.) water, while the mice in the TLPL and rhG-CSF groups were administered with 100 mg kg⁻¹ TLPL and 22.5 μg kg⁻¹ rhG-CSF for 3 weeks.

2.5 Body weight and organ indices

The body and organ weights of the mice were measured using an electronic balance. At the end of the experiment, the organs were immediately removed and weighed. The organ indices (%) were calculated according to the following formula:

Organ index (%) = organ weight (mg)/body weight (g) × 100%

2.6 H&E staining

Dissected femurs were fixed in 4% paraformaldehyde for 1 day and then decalcified in daily refreshed 0.5 M EDTA for 1 week. Fixed tissues were dehydrated through a series of graded ethanol baths and then embedded in paraffin. Paraffin-embedded blocks were cut into 5 μm thick sections and stained with hematoxylin and eosin (H&E).

2.7 Peripheral blood cell counts

Blood samples were collected with an EDTA-coated tube and the peripheral blood was directly analyzed using a 3-part differential veterinary hematology analyzer/CBC test machine.

2.8 ELISA assay

The levels of cytokines G-CSF (Shanghai Enzyme-linked Biotechnology Co., Ltd, YJ002290), EPO (Shanghai Enzyme-linked Biotechnology Co., Ltd, YJ002210), SCF (Shanghai Enzyme-linked Biotechnology Co., Ltd, YJ063287), PF4 (Shanghai Enzyme-linked Biotechnology Co., Ltd, YJ037257), TNF-α (NeoBioscience Technology Co., Ltd, EMC102a.96) and IFN-γ (NeoBioscience Technology Co., Ltd, EMC101g.96) in the serum were analyzed using ELISA kits according to the manufacturer’s protocol.

2.9 Detection of intestinal oxidative stress levels

Tissue homogenates were prepared from mouse intestinal tissues and assayed for SOD (Suzhou Grise Biotechnology Co. Ltd, G0101), MDA (Beyotime Biotechnology Co., Ltd, S0131S), and FRAP (Beyotime Biotechnology Co., Ltd, S0116) according to the manufacturer's protocol.

2.10 Lactate concentration analysis

Serum was obtained from retro-orbital plexus bleeding. The lactate concentration in the serum was measured using a lactate assay kit (Nanjing Jiancheng Bioengineering Institute, A019-2-1) as per the manufacturer's instructions. Absorbance was measured at 570 nm using a spectrophotometric microplate reader (Bio-Rad).

2.11 Bone marrow cell count assay

The intact femur was removed from one side of the mouse and soft tissue was removed. 1 mL PBS was absorbed with a syringe and inserted into the femur, allowing cells from the bone marrow to be flushed into a sterile centrifuge tube. After removing the red blood cells, the bone marrow cell suspension was filtered into a single-cell suspension using a 200-mesh filter. Appropriate amount of diluted cell suspension was absorbed and dropped into a cell counting plate for observation and counting under an optical microscope.

2.12 Flow cytometry for bone marrow populations

Bone marrow was obtained by flushing it out from the leg bones. The following antibody combination was used. PerCP-conjugated anti-mouse CD45 (BioLegend, 103129), APC-conjugated anti-mouse CD19 (BioLegend, 115511), FITC-conjugated anti-mouse Lineage Cocktail (biosciences, 97-7770-T100), PE-conjugated anti-mouse Sca-1 (BioLegend, 108107), APC-conjugated anti-mouse c-kit (BioLegend, 135108). Staining of the antibodies followed the protocol of the products. Samples after all staining were submitted for flow cytometry analysis using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences).

2.13 Real-time PCR

RNA was extracted using TRIzol from the total bone marrow (BM) cells isolated from the femur and tibia. Thereafter, the RNA was converted to cDNA using HiScript QRT SuperMix (Vazyme, R123-01). The cDNA was then used as the template for real-time qPCR conducted using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711-02) on a QuantStudio 3 real-time qPCR system (Applied Biosystems). The primer sequences used for real-time qPCR were as follows: Gpr81, 5′-GGCTGAGAAAAGCGGTATAGA-3′ and 5′-TCGTTAACTCTCTCCGAGCTAGA-3′; Ifitm3, 5′-TTCAGTGCTGCCTTTGCTC-3′ and 5′-CCTTGATTCTTTCGTAGTTTGGGG-3′; Stat1, 5′-CTGAATATTTCCCTCCTGGG-3′ and 5′-TCCCGTACAGATGTCCATGAT-3′; and 18S, 5′-GTAACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′.

2.14 Immunohistochemistry analysis

Immunohistological staining (IHC) of formalin-fixed paraffin-embedded tissue sections was performed using polyclonal antibodies to MyD88 (Servicebio, GB111554, dilution 1 : 200) and NOD1 (OriGene, R5579, dilution 1 : 200). For this purpose, 4 μm tissue sections were deparaffinised and heated in a water bath for 20 min. Then, the slides were immersed in a methanol solution of 3% hydrogen peroxide for 10 min. After washing with PBS, the slides were incubated with primary and secondary antibodies at room temperature in a humid and dark place. The slides were stained with a substrate chromogenic solution called 3,3′-diaminobenzidine tetrahydrochloride (DAB) for 5 min. The stain was restained with hematoxylin for 30 s and washed with water. The slides were then mounted for study under a microscope.

2.15 16S rDNA sequencing analysis

Fresh colon stool samples frozen in liquid nitrogen were submitted to Shanghai Majorbio Biotechnology Co., Ltd (Shanghai, China) for 16S rRNA sequencing analysis.

2.16 Statistical analysis

All experiments were repeated three times and all data results were processed using IBM SPSS Statistics Version 20.0 and are expressed as mean ± standard deviation. Significant differences were analyzed using Tukey's post hoc test comparison. Different letters (a–f) in the bars were considered to be statistical significance (Fig. 7G).

3 Results and discussion

3.1 Structural characterization of the TLPL

Fig. 1 shows the structural characterization of the TLPL. The molecular weight of the TLPL was 111 kDa as shown in the HPGPC chromatogram (Fig. 1A). It was composed of glucose (32.60%), galactose (25.30%), mannose (12.60%), rhamnose (8.10%), arabinose (7.50%), fucose (3.70%), and glucuronide (7.50%) (Fig. 1B). The ATR-FTIR spectrum in Fig. 1C revealed that the TLPL had the basic characteristics of polysaccharides. O–H tensile vibration at 3208 cm⁻¹, C–H tensile vibration at 2934 cm⁻¹, and C–O–H tensile vibration at 1079 cm⁻¹ were shown. The characteristic absorption peak of saccharide molecular vibration is in the region of 750–950 cm⁻¹, and the measurement result has a absorption peak at 864.4 cm⁻¹, which is presumed to be characteristic of α-epimer.²⁷Fig. 1D is the UV-Vis spectrum, which shows no significant absorption peaks at both 260 and 280 nm, indicating that the major proteins and nucleic acids were removed by the three-phase partitioning. Fig. 1E shows the conformation of the TLPL analysed using the Congo Red experiment, from which it can be seen that the wavelengths of the maximum absorption peaks of the TLPL in weak alkaline solution are all blue-shifted compared to the Congo Red control samples, indicating that the molecular structure of the TLPL did not contain a triple-helix conformation. The above detected structure of TLPL was quite different from the structure of the dried longan polysaccharide reported by Lan et al. (2021), which may be due to the different extraction methods.²⁸

Fig. 1 Structural characterization of the TLPL. (A) HPGPC diagram, (B) Ion Chromatography diagram detection, (C) ATR-FTIR spectrum, (D) UV detection, and (E) Congo Red assay.

3.2 TLPL restores body mass and ameliorates organ damage in mice

In this study, CTX was employed to establish a myelosuppression mice model, with the aim of evaluating the effect of TLPL on myelosuppression (Fig. 2A). During the animal modelling period, notable physiological changes were observed in the mice; they exhibited significant reductions in vigor and food intake, alongside severe weight loss and noticeable hair loss. These symptoms are indicative of systemic stress and compromised health status due to CTX administration. After 3 weeks of TLPL treatment, body weight exhibited a remarkable increase (p < 0.05) compared with the MC group (Fig. 2B). Moreover, the organ index can reflect the physiological and pathological status of organs in different treatment groups of mice, and evaluate the intervention effect of the TLPL. As expected, CTX induced severe immune organ damage in the mice, including splenomegaly and thymic atrophy, whereas the TLPL had a protective effect on the damaged organs. The splenic indices of mice in the MC, NC, TLPL and rhG-CSF groups were 9.53%, 3.33%, 6.49% and 6.92%, respectively, whereas the thymic indices were 0.98%, 1.78%, 2.08% and 2.16%, respectively (Fig. 1C). The spleen and thymus are important haematopoietic-related organs in addition to bone marrow.²⁹ CTX-induced bone marrow haematopoietic dysfunction leads not only to diminished blood cell production but also results in hypersplenism due to splenomegaly—this condition can significantly reduce platelet counts among other cellular components vital for hemostasis and overall health maintenance within an organism's circulatory system. The destruction of the thymus affects the development of T-lymphocytes, which reduces the immune function of the organism. These results were in agreement with the results reported by Li et al. (2023) for polysaccharides from Gastrodia elata.³⁰ In conclusion, TLPL can effectively promote the regeneration and development of damaged immune cells and improve immunity.

Fig. 2 Mice orally dosed with TLPL were protected from CTX-induced myelosuppression. (A) Experimental scheme, (B) TLPL prevented the weight loss of mice with myelosuppression, (C) organ index in different groups, (D–J) peripheral blood count (WBC: p < 0.05, df = 3, F = 26.127; LYMP: p < 0.05, df = 3, F = 19.375; Gran: p < 0.05, df = 3, F = 9.115; Mo: p > 0.05, df = 3, F = 2.363; PLT: p < 0.05, df = 3, F = 12.673; HGB: p < 0.05, df = 3, F = 20.335 and RBC: p > 0.05, df = 3, F = 0.381). Each dot represents an individual mouse. n = 6–10 mice per group. Bars are ± SD. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test comparison. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. the NC group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the MC group. MC, model control group; WBC, white blood cell; LYMP, lymphocytes; Gran, granulocytes; Mo, monocytes; PLT, platelets; HGB, hemoglobin; RBC, red blood cell; df, degree of freedom and F, F value.

3.3 TLPL promotes recovery of peripheral blood cell counts

To understand the effect of TLPL on the restoration of haematopoiesis in myelosuppressed mice, peripheral blood cell counts were measured to indirectly reflect the haematopoietic capacity of the bone marrow. rhG-CSF was used as a positive control, which is widely used in the clinic to trigger haematopoietic stem cell (HPCs) and restore haematopoiesis. The results of the study of haematological parameters in different groups of mice are presented in Fig. 2D–J, including the levels of white blood cells (WBC), lymphocytes (LYMP), neutrophils (Gran), monocytes (Mo), platelets (PLT), haemoglobin (HGB) and red blood cells (RBC). As expected, the number of blood cells, except Mo and RBC, were significantly reduced in the MC group, consistent with the manifestation of early myelosuppression. For WBC levels, the MC, NC, TLPL, and rhG-CSF groups had 0.72 ± 0.35 10⁹ L⁻¹, 5.58 ± 0.83 10⁹ L⁻¹, 3.18 ± 1.711 10⁹ L⁻¹, and 3.48 ± 1.65 10⁹ L⁻¹, respectively. TLPL and rhG-CSF treatments significantly elevated the WBC levels. Meanwhile, the effect of TLPL on the production of Mo, Gran and LYMP was similar to the stimulation of WBC. It was suggested that Mo, Gran and LYMP are all WBC phenotypes, which are important for the maintenance of the normal function of the immune system.³¹ The platelet (PLT) concentration of the TLPL group was 1238.1 ± 164.41 10⁹ L⁻¹, which was 2.28 times and 1.17 times higher than that of the MC and NC, respectively. The HGB concentrations in the MC, NC, TLPL and rhG-CSF groups were 116.75 ± 7.42, 162.90 ± 17.38, 135.10 ± 4.42, and 135.10 ± 4.42 g L⁻¹, respectively. Compared with NC, the HGB in MC decreased by 28.33%, and the HGB in the TLPL and rhG-CSF groups rebounded to 82.93% and 85.51% that of the NC group, respectively. However, CTX did not have a significant effect on the RBC counts in the four groups, which may be related to the inconsistent effects of CTX on different blood cell life cycles, as CTX is primarily used to model myelosuppression with leukocytes and platelets as the main observations.^32,33

3.4 TLPL protected the homeostasis of the BM haematopoietic microenvironment

Fig. 3A shows the effect of TLPL on the histopathological status of the femurs using the H&E staining method. In the MC group, haematopoietic cells in the femoral cavity were significantly reduced and the haematopoietic area was significantly decreased, while adipocytes were significantly increased and filled up a large amount of the area of the femoral cavity (red arrow). The TLPL group significantly restored the phenomenon of inflammatory infiltration of the bone marrow cells, and increased the proportion of the cells. The same results were confirmed by counting the nucleated cells in the femur and humerus of different groups (Fig. 3B and C), and TLPL significantly increased the number of nucleated cells in the femur and tibia of the myelosuppressed mice (p < 0.05). The numbers of bone marrow luminal cells in the MC, NC, TLPL and rhG-CSF groups were 0.62 × 10⁷, 3.78 × 10⁷, 1.70 × 10⁷ and 1.85 × 10⁷, respectively. Fig. 3D and E show the proportion of B lymphocytes (CD45⁺CD19⁺), haematopoietic stem cells (HSCs, lin⁻sca-1⁺c-kit⁺) and haematopoietic progenitor cells (HPCs, lin⁻sca-1⁺c-kit⁺) in the bone marrow cells of mice in different groups. As depicted in Fig. 3D, the amounts of B lymphocytes in the MC, NC, TLPL and rhG-CSF groups were 4.55%, 28.10%, 11.10% and 11.10% respectively. The HPC amounts in the MC, NC, TLPL and rhG-CSF groups were 2.22%, 11.01%, 5.16%, and 4.23%, respectively, and the HSC amounts in the different groups were 0.43%, 2.15%, 1.40% and 1.76%, respectively. After TLPL intervention, the amount of HSCs increased by 2.94% and the amount of HPCs increased by 0.97% compared to the results of the MC group.

Fig. 3 Effects of TLPL on the haematopoietic microenvironment in BM. (A) Histopathological observation (H&E staining), (B) the color of the bone marrow cells in different groups, (C) the amount of nucleated bone marrow cells in different groups, p < 0.05, df = 3, F = 36.123, (D) B lymphocytes and (E) HSCs and HPCs in the different groups. Each dot represents an individual mouse. n = 8 mice per group. Bars are ± SD. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test comparison. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. the NC group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the MC group.

From birth, the bone marrow becomes the site of haematopoietic stem cell proliferation and differentiation and maintains lifelong haematopoiesis in the organism. B lymphocytes play an important role in the immune response of the organism, and TLPL promotes the production of B lymphocytes in bone marrow cells, which may be conducive for B lymphocytes to promote the proliferation and differentiation of bone marrow stem cells and to regulate the migration and localisation of haematopoietic stem cells, which are involved in haematopoiesis.³⁴ Meanwhile, TLPL also promotes the haematopoietic stem cells (HSCs), and haematopoietic progenitor cells (HPCs). HSCs are the initial progenitors of myeloid and lymphoid stem cells in the bone marrow, which maintain the normal function of the entire haematopoietic system.³⁵ They are able to regenerate and differentiate pluripotently, and can differentiate into differentiation cells. In recent years, the prominent effects of plant polysaccharides in improving haematopoietic dysfunction have been demonstrated, and similar to TLPL, digitonin polysaccharides promote the recovery of bone marrow granulosa progenitor cells after the action of cyclophosphamide.⁶

3.5 TLPL improved haematopoietic cytokine secretion

Cytokines play an important role in regulating the myeloid haematopoietic response, and the effects of TLPL on four positively regulated cytokines and two negatively regulated cytokines are shown in Fig. 4. The observed G-CSF, PF4, EPO, and SCF indices in positive haematopoietic cytokines suggested that TLPL improved the secretory proteins that regulate the cell function, thereby improving myelopathic inhibition. After 21 days of TLPL gavage, the secretion levels of all four cytokines were similarly increased to 636.58 ± 60.99 pg mL⁻¹, 9.67 ± 0.46 ng mL⁻¹, 35.35 ± 1.09 IU L⁻¹, and 308.73 ± 29.12 pg mL⁻¹, respectively, which were 2.44 times, 1.57 times, 1.13 times, and 1.14 times that of the MC group. Yet, the serum levels of TNF-α and IFN-γ were significantly increased in the MC group, which were 557.66 ± 38.29 pg mL⁻¹ and 52.24 ± 5.35 pg mL⁻¹, respectively. However, TLPL decreased the secretion of TNF-α and IFN-γ by 21.68% and 12.85% compared with that of the MC group, which significantly suppressed the production of cellular inflammatory cytokine (p < 0.05).

Fig. 4 The haematopoietic activity of TLPL. (A–F) Serum cytokine secretion tests by ELISA assay (G-CSF: p < 0.05, df = 3, F = 100.310; PF4: p < 0.05, df = 3, F = 7.829; EPO: p < 0.05, df = 3, F = 5.203; SCF: p < 0.05, df = 3, F = 32.960; TNF-α: p < 0.05, df = 3, F = 16.769; IFN-γ: p < 0.05, df = 3, F = 24.617). Each dot represents an individual mouse. n = 6–10 mice per group. Bars are ± SD. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test comparison. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. the NC group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the MC group. G-CSF, granulocyte-colony stimulating factor; PF4, platelet factor 4; EPO, erythropoietin; SCF, stem cell factor; TNF-α, tumor necrosis factor-α; IFN-γ, interleukin-γ.

The haematopoietic process of bone marrow is under the mutual regulation of many kinds of blood cells, in which cytokines regulate cell survival, proliferation, differentiation and apoptosis by binding to their specific receptors, and the secretion of these cytokines directly affects the haematopoietic process. Among them, TLPL had the most significant effect on the promotion of G-CSF, a pleiotropic cytokine that can stimulate the proliferation and differentiation of stem cells in the bone marrow, promote the balance of cytokines in the haematopoietic microenvironment, promote the bone marrow homing of HSCs, regulate the function of the haematopoietic system, and at the same time stimulate the intrinsic immune response.³⁶ Additionally, EPO can inhibit apoptosis (programmed cell death) of erythrocyte precursors, ensuring their survival and maturation into functional red blood cells. Compared with the other three haematopoietic cytokines, the inhibitory effect of cyclophosphamide on EPO is weaker, and the results are consistent with the indicators of RBCs in the peripheral blood of mice in all groups. TNF-α and IFN-γ can be considered as partial negative modulators of haematopoiesis; elevated concentrations of TNF-α can trigger apoptosis in haematopoietic cells, such as granulocytes and erythrocyte precursor cells, resulting in their reduced numbers and consequently impacting haematopoiesis.³⁷ Additionally, IFN-γ exerts an inhibitory effect on the proliferation and differentiation of bone marrow stem cells, thereby influencing haematopoietic processes. Notably, it specifically affects the self-renewal and differentiation potential of pluripotent stem cells.^38,39 These results significantly revealed that TLPL may have the ability to inhibit the inflammatory response triggered by myelosuppression, which effectively prevents cell damage or death in the bone marrow by reducing the release of inflammatory mediators and finely regulating the secretion of haematopoietic cytokines, thus helping the body to restore haematopoietic function. This finding coincides with previous research results and further solidifies the potential of TLPL in related fields.⁴⁰

3.6 TLPL ameliorated intestinal oxidative stress levels in mice

The effects of different treatment groups on FRAP capacity and SOD activity in the intestines of mice are shown in Fig. 5A and B. Compared with the NC group, the intestinal FRAP and SOD capacities in the MC group were significantly reduced (p < 0.001) to 0.46 ± 0.02 mM and 20.93 ± 2.02 U mg⁻¹ prot, which were 23.33% and 31.46% lower, respectively. In contrast, these capacities were elevated when gavaged with TLPL and rhG-CSF. TLPL increased the FRAP capacity of myelosuppressed mice by 7.90% and the SOD activity by 14.58% compared with the MC group. The MDA content is shown in Fig. 5C. In the MC group, MDA was 0.055 ± 0.001 μmol mg⁻¹, which was significantly higher than that in the NC group. Notably, TLPL and rhG-CSF results showed significantly reduced contents of 0.035 ± 0.002 μmol mg⁻¹ and 0.031 ± 0.001 μmol mg⁻¹ (p < 0.001), respectively. The above results showed that TLPL could reduce the oxidative stress capacity of the intestine and maintain intestinal health.

Fig. 5 Effects of TLPL on the intestinal oxidative stress levels. (A) FRAP: p < 0.05, df = 3, F = 55.780, (B) SOD activity: p < 0.05, df = 3, F = 40.823, (C) MDA content: p < 0.05, df = 3, F = 95.685. Each dot represents an individual mouse. n = 8–10 mice per group. Bars are ± SD. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test comparison. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. the NC group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the MC group.

It is important to acknowledge that gut health is important in regulating the environmental homeostasis of the bone marrow. Interaction systems exist between the gut and bone marrow, which are interconnected through a complex signalling network.^21,41 Stimulation of mice with CTX leads to excessive oxidative stress in the body, resulting in the production of large amounts of oxygen free radicals and other oxidation products, which can damage colonic tissues to varying degrees. In this study, TLPL increased the presence of the antioxidant enzyme (SOD) in the gut, allowing SOD to catalyse the dismutation (or portioning) of superoxide radicals into ordinary molecular oxygen, reducing the production of lipid peroxides (MDA) and protecting membrane phospholipids from oxidative damage, which may help to improve intestinal mucosal immune function.

3.7 TLPL altered the microbial taxonomic profile and metabolic activity of myelosuppressed mice

As shown in Fig. 6A and B, gut microbiota characteristics at the phylum level and genus level were changed significantly during myelosuppression. At the phylum level, Bacteroides, Firmicutes, and Actinobacteriota were the dominant flora in four groups. CTX significantly decreased the relative abundance of Actinobacteriota, Proteobacteria, and Desulfobacterota (p < 0.05) and significantly increased the relative abundance of Bacteroides (p < 0.05) compared with the NC group (Fig. 6A). Interestingly, TLPL remodelled the structure of the gut microbiota, suggesting that administration of plant-derived polysaccharides may be associated with health management, consistent with a large number of previous studies.^42,43 Compared to the MC group, TLPL and rhG-CSF significantly increased the relative abundance of Firmicutes, Actinobacteriota, and Proteobacteria (p < 0.05) and decreased the relative abundance of Bacteroides. At the genus level, TLPL and rhG-CSF increased the CTX-induced reductions in the relative abundance of the beneficial bacteria Staphylococcus, Lactobacillus, Corynebacterium, and Enterorhabdus. Compared with the MC group, Lactobacillus, Corynebacterium, and Glutamicibacter were significantly increased in the TLPL group, with relative abundances of 24.79%, 7.59%, and 4.83%, respectively, and all of them are important beneficial bacteria in the organism (Fig. 6B). In Fig. 6C, information for a total of 67 species is shown, and significant differences between groups were found at the phylum, order, family and genus levels from the branching evolution diagram (p < 0.05), of which 24 microbial groups in the TLPL group had significantly higher relative abundance than the other groups (p < 0.05). To further identify bacterial taxa with differential abundances, linear discriminant analysis effect size (LEfSe) was used. Fig. 6D shows the obtained results through the data dimensionality reduction of the metrics. According to the order of the linear discriminant analysis (LDA) score, the top 5 categories were o__Lactobacillales, c__Bacilli, o__Micrococcales, g__unclassified__f__Rhodobacteraceae, and g__Acinetobacter. Importantly, in the above abundance analysis data, special attention was paid to the Lactobacillus with increased abundance in the TLPL group. Lactobacillus is associated with potential health benefits due to regulation of glucose and lipid metabolisms, as well as being a typical probiotic of the Firmicutes phylum that can maintain the intestinal barrier and effectively inhibit intestinal infections through the production of short-chain fatty acids and lactate.⁴⁴ These results were similar to Wan et al. (2023),²¹ which showed a positive correlation between Lactobacillus and haematopoietic efficacy. It was concluded that the γ-aminobutyric acid (GABA) and lactate, metabolites of Lactobacillus, regulated haematopoietic stem cells and haematopoietic progenitors; meanwhile, lactate promotes the proliferation of mesenchymal stem cells (MSC) and accelerates haematopoietic stem cells.²¹

Fig. 6 Effects of TLPL on the intestinal microenvironment, gut microbiota abundance and function. (A) Phylum levels, (B) genus levels, (C) LEfSe taxonomic cladogram, (D) the LDA scores (LDA greater than 3.5) and (E) prediction of microbial community function. n = 6 mice per group.

Of note, the physiological effects and health benefits resulting from utilization of prebiotics may be explained not only by changes in gut microbiota abundance, but also by changes in microbiota functionality or metabolism. Fig. 6E shows the predicted metabolic pathways in the MetaCyc database (the annotated table of metabolic pathways is shown in the ESI as Table S1†). In the MC group, the abundances of the aerobic respiration I (cytochrome c) metabolic pathway and guanosine nucleotide ab initio biosynthesis superpathway I were significantly decreased (PWY-3781 and PWY-7288). However, TLPL increased the functional abundance of these metabolic pathways. For the MC group, this may be due to tissue hypoxia resulting from myelosuppression, causing an inadequate supply of tissue oxygen, as well as restricted redox reactions, leading to impaired electron transfer between the cytochrome system (e.g. cytochrome c) and cytochrome oxidase.^45,46 Chronic profound hypoxia may alter the diversity of the intestinal microbiota, leading to the accumulation of intestinal microbial-derived metabolites, which may significantly affect host homeostasis. The presence of Lactobacillus may ameliorate this adverse reaction. Lactobacillus is one of the most important probiotic bacteria responsible for the metabolism of lactose and D-galactose in the intestinal tract.⁴⁷ Xing et al., (2018) suggested that supplementation of chronic hypoxic rats with Lactobacillus reduces the accumulation of D-galactose in the bone marrow and prevents the accumulation of D-galactose from having a toxic effect on the cells, which would allow premature senescence of bone marrow mesenchymal stem cells.⁴⁷ Combining the results from the two-part analysis of the microbiota, including abundance and function, it is reasonable to speculate that the high abundance of Lactobacillus in the TLPL group may be involved in the metabolism of D-galactose in the intestinal tract, affecting the communication between the intestinal tract and the bone marrow, and thus improving the bone marrow microenvironmental homeostasis in which the haemopoietic cells live.

Taken together, TLPL supplementation may restore the health of the organism by modulating the structure of the gut microbiota, promoting the enrichment of beneficial bacteria such as Lactobacillus. At the same time, Lactobacillus exerts special metabolic functions, such as increasing D-galactose metabolism or the aerobic respiration I (cytochrome c) metabolic pathway to help restore the haematopoietic environment.

3.8 TLPL effects on bone marrow haemopoiesis are associated with intestinal metabolites

In recent years, three mechanisms related to the regulation of host haematopoiesis by gut microbiota have been reported: (1) the gut microbiota metabolite NOD1L promotes haematopoiesis through activation of type I IFN and up-regulation of the Stat1, Ifitm3 signalling pathway;⁴⁸ (2) Lipopolysaccharide (LPS) secreted by intestinal secretion promotes myeloid cell formation through the blood circulation with the downstream pathway of MyD88 on haematopoietic progenitor cells, which then contributes to homeostatic haematopoiesis; and (3) lactate, a metabolite of gut microbiota, reaches the bone marrow through blood circulation and stimulates SCF secretion from LepR+ stromal cells around the bone marrow sinus in a Gpr81 signalling-dependent manner, further promoting myeloid differentiation²² (Fig. 7A).

Fig. 7 Mechanism of TLPL to ameliorate haematopoietic activity in myelosuppressed mice. (A) Schematic representation of relevant mechanisms. (B) Lysozyme activity, p < 0.05, df = 3, F = 5.029. (C) NOD1 expression on bone marrow cells. (D) LPS content, p < 0.05, df = 3, F = 3.810. (E) MyD88 expression on bone marrow cells. (F) Serum lactate content, p < 0.05, df = 3, F = 28.893. (G) Gene expression on bone marrow cells test by RT-qpcr (Ifitm3, Stat1 and Gpr81). (H) Mechanism of the haematopoietic activity of TLPL. Each dot represents an individual mouse. n = 6–10 mice per group. Bars are ± SD. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test comparison. ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 vs. the NC group. * p < 0.05, ** p < 0.01 and *** p < 0.001 vs. the MC group.

To gain insights into the haematopoiesis mechanism of TLPL-responsive microbes and their metabolite, qPCR and IHC were performed to inspect individually. Since lysozyme can release NOD1L from bacteria by hydrolysing bacterial peptidoglycan in the intestinal lumen,⁴⁹ the intestinal NOD1L level was first analysed indirectly by determining the level of lysozyme in the intestinal tract, and the results are shown in Fig. 7B. TLPL significantly increased the level of lysozyme in the intestinal tracts of the myelosuppressed mice. However, the expression level of the NOD1 protein in the myeloid cells was not significantly different from that of the MC group (Fig. 7C, no positive expression in all four groups), and at the same time, the corresponding gene expression levels of Stat1 and Ifitm3 were not up-regulated, thus excluding the possibility that TLPL promotes haematopoiesis in the organism by mechanism 1. Afterwards, LPS levels in the intestine were shown in Fig. 7D. TLPL eliminated the cyclophosphamide-induced LPS elevation. LPS has a wide range of biological activities and can regulate the immune system in many ways (stimulation of haematopoiesis), but at the same time, LPS is an endotoxin released by gram-negative bacteria, and a large amount of LPS activates monocytes and releases cytokines to initiate systemic inflammation.⁵⁰ The release of cytokines can initiate a systemic inflammatory response, leading to intestinal inflammation. Fig. 7E shows the MyD88 expression in bone marrow, and the protein expression level of the MC group was significantly higher than that of the other three groups, which is the opposite of mechanism 2. Finally, mechanism 3 with lactate as the signaling medium was explored. Intestinal metabolites can be transported through the portal vein into the peripheral circulation to act on peripheral tissues, and despite the low levels of metabolites in the peripheral circulation, they can still act as signalling molecules to participate in the host's biological processes. Interestingly, lactate, a common intestinal metabolite from Lactobacilli, was significantly increased in the serum in the TLPL group (p < 0.05) with 20.00 ± 2.33 mmol L⁻¹ (Fig. 7F). Since TLPL treatment suppressed intestinal oxidative stress induced by cyclophosphamide while reducing the level of inflammation in the tissues, the possibility that it was due to lactate production by cellular glycolysis in the hypoxic environment of the tissues caused by inflammation was ruled out. Therefore, TLPL may modulate the abundance of microbiota in mice and promote the proliferation of lactate-producing microorganisms, such as Lactobacillus, thereby increasing the level of lactate, and helping communicate with bone marrow cells through blood circulation. Moreover, the gene expression of Gpr81 in BM further supported the conclusion that lactate may bind to corresponding receptors through blood circulation, suggesting that the haematopoietic function of TLPL is a potential result of the interaction with gut microbiota.

Together, the mechanism underlying the haematopoietic role of TLPL may involve the gut microbiota. TLPL was beneficial to the proliferation of Lactobacillus and further produces lactate under intestinal fermentation. After entering the blood circulatory system, lactate may act as a signalling molecule for achieving long-distance communication between gut microbes and bone marrow haematopoietic cells, stimulating bone marrow cells in a Gpr81 signal-dependent manner, promoting the production of haematopoietic cytokines, such as SCF, and helping to restore peripheral blood counts and reverse myelosuppression in the organism (Fig. 7H).

4 Conclusions

In this study, the effect of TLPL on the improvement of bone marrow haematopoiesis was assessed. In summary, TLPL supplementation effectively reduced weight loss, alleviated spleen, thymus and bone marrow cavity damage, and promoted the recovery of peripheral blood counts in myelosuppressed mice. Meanwhile, TLPL reduced intestinal oxidative stress, and changed the composition and function of gut microbiota. It was shown to promote the proliferation of Lactobacillus and enhance the aerobic respiratory metabolic activity of the flora. In turn, this promoted the fermentation of TLPL by Lactobacillus to produce lactate. After entering the blood circulation, lactate acts as a signal molecule for long-distance communication between intestinal microorganisms and bone marrow haematopoietic cells, and stimulates bone marrow cells in a Gpr81 signal-dependent manner, promoting the production of haematopoietic cytokines. These findings provide an explanation for the haematopoietic mechanism of dried longan, and a theoretical basis for further rational application.

Author contributions

Shiai Zeng: methodology, investigation, formal analysis, writing – original draft. Lan Gao: methodology, investigation. Kai Wang: methodology, writing – review & editing. Xuwei Liu: formal analysis, data curation. Zhuoyan Hu: project administration, funding acquisition. Lei Zhao: conceptualization, methodology, writing – review & editing, project administration, funding acquisition.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors declare no competing financial interest.

Acknowledgements

The work was supported by the Guangzhou Key R&D Programme (2024B03J1307), and the Natural Science Foundation of Guangdong Province (2023A1515012599 and 2024A1515012220).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4fo03758h

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