Isolation of exosome-like nanovesicles from C. roseus

C. roseus-derived nanovesicles were separated by differential ultracentrifugation after being crushed and juiced from fresh C. roseus. It was found that C. roseus-derived nanovesicles contained three nanovesicles with different particle sizes according to experimental results from transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis (Additional file 1: Fig. S1B and C). To avoid possible interference between different plant tissues, nanovesicles were recovered from leaves (Additional file 1: Fig. S1D), stems (Additional file 1: Fig. S1E) and flowers (Additional file 1: Fig. S1F). These nanovesicles differ in morphology and size. Among them, leaves-derived nanovesicles with more homogeneous morphology and exosome-like characteristics were selected for the study.

We speculated that the juicing process can cause the plant to release large amounts of water-soluble pigments and also damaged the plant cells making the organelles spill out (especially chloroplasts, etc.). In order to obtain more purified extracellular vesicles, the extraction method was modified according to Ref [32] (Additional file 1: Fig. S1G and H). In brief, the leaves of C. roseus were cleaned, digested with pectinase and cellulase, and then centrifuged to remove protoplasts. The supernatant was concentrated using hollow fibers, and then CLDENs were extracted using sucrose buffer-ultracentrifugation (Fig. 1A). Extracellular vesicles isolated using the aforementioned technique were suspended in phosphate buffer solution and kept at −80 °C until use.

Fig. 1

Isolation and physicochemical characterization of CLDENs. A Schematic diagram of the separation process of CLDENs. B TEM analysis of CLDENs. Scale bar = 100 nm. C DLS was used to analyze the particle size distribution of CLDENs. D A nanoflow analyzer picked up the CLDENs particle size. E Analysis of CLDENs’ zeta surface charge. F,G Particle size changes of CLDENs in enzyme (F) and surfactant-containing (G) environments. H Stability of CLDENs in different pH environments. Particle size changes of CLDENs in pH = 6.92 (a), pH = 13.58 (b) and pH = 1.83 (c) environments. TEM imagines of CLDENs in pH = 6.92 (d), pH = 13.58 (e) and pH = 1.83 (f) environments. Scale bar = 200 nm. The white triangle marked the two fused vesicles

Physicochemical characterization of CLDENs

Particle size analysis on CLDENs was tested by TEM within 48 h after the extraction. It was demonstrated that CLDENs had a rounded hollow vesicle shape and had particle size between 50 and 100 nm (Fig. 1B). DLS analysis indicated that the particle size of CLDENs in aqueous solution was 141.7 nm (Fig. 1C). We noticed that the difference between DLS analysis and TEM analysis in the detection of exosome particle size was a common phenomenon [33]. Therefore, we additionally used nano-flowmeter to complement the particle size of CLDENs and found that the particle size of CLDENs were 75.51 ± 10.19 nm (Fig. 1D).

Zeta potential is a significant characteristic that can be used to assess the stability of nanoparticle colloids. In general, nanoparticles with zeta potential values between -30 mV ~ + 30 mV are considered to have better stability [34]. It was found that CLDENs had a zeta potential of -21.8 mV in aqueous solution, indicating that the vesicles were stable (Fig. 1E).

Multiple methodologies were used to evaluate CLDENs’ stability. CLDENs were exposed to 0.02% Triton X-100 and 0.1% SDS for 30 min. The original single particle size peak of CLDENs split, indicating the destruction of vesicles (Fig. 1F). After being digested for 30 min with RNase (3 μg/ml), DNase (6 μg/ml), and proteinase K (100 μg/ml), the particle size distribution of CLDENs did not alter significantly, which indicated that the vesicles did not suffer damage (Fig. 1G). When immersed in sodium hydroxide solution and hydrochloric acid solution for 30 min, the particle size peak of CLDENs was not split but displaced (Fig. 1Ha-c). The variation of biomaterials in different pH environments is an important method to evaluate their stability [35]. To determine whether the rupture of CLDENs occurred, we observed the morphology of CLDENs under TEM after acid or alkali treatment. Compared with the control group (Fig. 1Hd), some nanovesicles expanded in size in an alkaline environment (Fig. 1He). This phenomenon was more obvious in an acidic environment, and the volume of vesicles changed from about 70 nm to more than 200 nm (Fig. 1Hf). As an acidic organelle, the change of pH environment can affect the membrane exchange and membrane fusion of MVB [36, 37]. Interestingly, we also observed the vesicle fusion of CLDENs in an acidic environment (Fig. 1Hf, Additional file 1: Fig. S2A and B). Even though, CLDENs still maintained a membrane vesicle-like structure without significant rupture even in acidic or alkaline environments. These data indicated that surfactants were able to break down CLDENs. CLDENs were resistant to acidic and alkaline environments, as well as the digestion of DNase, RNase, and protease.

Biocompatibility and biodistribution of CLDENs

CLDENs were labeled with red fluorescent dye PKH26 and were co-incubated with RAW 264.7 cells. It was showed that CLDENs were taken up by macrophages and deposited primarily in the cell cytoplasm. The red fluorescent signal gradually increased in RAW264.7 cells by time prolonged (Fig. 2A). Additionally, as the co-incubation period increased, the morphology of the RAW264.7 cells gradually changed, primarily as evidenced by an increase in cell volume and the number of pseudopodia (Marked by the white triangle in Fig. 2A).

Fig. 2

Biocompatibility of CLDENs. A CLDENs accumulated intracellularly over time after uptake. White triangles were used to mark RAW264.7 cells’ morphological changes after ingestion of CLDENs. Scale bar = 20 μm. B Cytotoxicity assay of CLDENs against multiple cell lines (n = 3). C Hemolysis test under different concentrations of CLDENs by using blood of sheep (n = 3). D Changes in the particle size distribution of CLDENs after incubation in PBS (a), stomach simulating fluid (b) and small intestine simulating fluid (c) at 37 °C

The MTT assay was used for detecting CLDENs’ cytotoxic effects on HIEC-6 cells, L-02 cells, RAW264.7 cells. Based on well-established guidelines ISO 10993.5, the materials for medical use were considered not cytotoxic when the cell viability rate was greater than 70% [38]. It appeared that the cell viability rate remained above 70% even at the highest concentration (1000 μg/ml) of CLDENs (Fig. 2B). These data suggested that CLDENs were relatively safe nano-vesicles.

Whole sheep blood was used for the evaluation of the hemolytic effect of CLDENs. As shown in Fig. 2C, no significant hemolysis was observed after incubating sheep blood with 0.05–8 mg/mL of CLDENs at 37 °C for 30 min. Compared with the negative control (PBS, which was regarded as having a 0% hemolysis rate) and the positive control (0.1% Triton X-100, which was regarded as having a 100% hemolysis rate), the hemolysis rate of CLDENs were less than 1%. These data showed that CLDENs possessed good biocompatibility.

Before observing the in vivo distribution of CLDENs, we examined the stability of CLDENs in an artificially simulated in vivo environment. The particle size distribution of CLDENs will not be affected when it was placed at 37 ℃ for 12 h (Fig. 2Da). Moreover, when CLDENs were placed in small intestine simulating fluid (Fig. 2Db) and stomach simulating fluid (Fig. 2Dc) at 37 °C, they could maintain stability for at least 12 h.

Then, we gave mice injections in the tail vein, intraperitoneal administration, and oral administration of 60 mg/kg of CLDENs, which were labeled with the near-infrared lipophilic fluorescent dye DIR. At regular intervals, animals were killed and their organs and tissues were taken to look for fluorescent signals.

Following oral treatment, only in the mice's abdominal region, the signs of CLDENs-DIR seen (Fig. 3Aa). After the animals were dissected, a minor fluorescent signal was seen in the liver, brain and lung (Additional file 1: Fig. S3Aa). Besides, a sustained, intense fluorescent signal was discovered in the mouse stomach which was above the instrument detection threshold and gradually waned after 12 h (Additional file 1: Fig. S3Ab). The CLDENs-DIR fluorescent signal did not expel from the stomach, even though the mice had free access to food and water. Combined with the results of in vitro experiments, we hypothesized that CLDENs may remain stable in gastric juice and have potent stomach absorption.

Fig. 3

Biodistribution of CLDENs. A Biodistribution of CLDENs in mice after oral administration (a), intraperitoneal injection (b), and tail vein injection (c). B Biodistribution of CLDENs in the organs after intraperitoneal injection administration. a. Fluorescent signals in the brain, heart, liver, spleen, thymus, lung and kidney. b. Fluorescent signals in the gastrointestinal tract

In the intraperitoneal injection group, CLDENs were found to be distributed in the immune organs. Fluorescent signals were observed in the thymus, spleen, pulmonary vasculature, liver, kidney (Fig. 3Ba) and the gastrointestinal system (Fig. 3Bb). In animals’ bodies, the fluorescent signal first appeared in the abdomen, and then in the testes, paws, and spread to the head over time (Fig. 3Ab), although no fluorescent signal was observed in the brain. It's interesting to note that some animals’ cervical lymphatic fraction also showed CLDENs-DIR signals (Additional file 1: Fig. S4).

Consistent with earlier reports, tail vein injection did not show more extensive systemic absorption than intraperitoneal injection [39]. CLDENs-DIR were primarily found in the animal's upper abdomen and extremities, except for the caudal injection site (Fig. 3Ac). Moreover, the tail vein injection group showed a much weaker fluorescent signal at the same scale. Only the liver and spleen of the animals' dissected organs contained CLDENs-DIR (Additional file 1: Fig. S3Ba). We hypothesized that CLDENs-DIR were quickly captured and processed by the liver and spleen after entering the circulatory system. Furthermore, robust fluorescent signals in the liver continued to be seen for 48 h.

To sum up, different modes of administration can cause CLDENs to exhibit very different biodistributions. Our data suggested that CLDENs may have excellent anti-gastric acid digestibility and can remain in the gastrointestinal tract for a long time after oral administration. Moreover, immune organ-targeted distribution can occur by intraperitoneal injection of CLDENs. CLDENs injected intravenously into the bloodstream may face interception and rapid clearance by the liver and spleen.

Compositional analysis of CLDENs

As a biological membrane vesicle, lipid makes up the majority of extracellular vesicles [40]. Non-targeted lipidomic was used to analyze the lipid composition of CLDENs. It was found that CLDENs contained mainly ether-phosphatidylcholines (EtherPC, ~ 16.55%), phosphatidylglycerols (PG, ~ 15.69%), phosphatidylinositol (PI, ~ 14.01%) and ether-phosphatidylglycerols (EtherPG, ~ 9.35%) (Fig. 4A and Additional file 1: Table S1). Exosomes are usually rich in saturated phospholipids, sphingomyelin, cholesterol, and phosphatidylserine [33, 41]. It was worth noting that CLDENs contained over 30% ether-phospholipids. Ether-phospholipids are a distinct type of phospholipid in which the sn-1 arm of the glycerol backbone is linked by an ether bond rather than an ester bond [42]. They play an important role in promoting the differentiation of neural cells [43], and the activation of neutrophils and macrophages [44, 45]. Moreover, ether-phospholipids have been reported to maintain membrane structure and integrity in nematode-derived exosomes, and participate in the host immune response [46]. The high content of ether-phospholipids may be responsible for the excellent stability exhibited by CLDENs. Also, these findings prompted us to speculate about CLDENs’ potential involvement in the immunomodulatory processes.

Fig. 4

Compositional analysis of CLDENs. A Lipidomic analysis of CLDENs. B Metabolomic analysis of CLDENs. C Molecular weights distribution of the identified proteins in the CLDENs group. D Volcano plot of proteomic analysis. E The number of differentially up-regulated and down-regulated expressed proteins. F, G Subcellular localization of differentially expressed proteins, up-regulated proteins (F) and down-regulated proteins (G)

Non-targeted metabolomic analysis was performed to identify compounds contained in CLDENs. The result showed that CLDENs mainly contained compounds such as amino acids, fatty acids and lipids, alkaloids, and carbohydrates (Fig. 4B and Additional file 1: Table S2). We paid special attention to the outstanding antitumor compounds contained in C. roseus, indole alkaloids. Vinpocetine was found in CLDENs, but not the well-known vinblastine, vincristine, amorphine, or vinblastine. It was reported that secondary metabolite enrichment was not exhibited all PDENs. Researchers found nanovesicles derived from grapefruit and ginger included naringin, naringenin [16], and shogaol [47], whereas those derived from orange lacked vitamin C and naringenin [48]. In this regard, we hypothesized that the types of compounds present in PDENs were influenced by the extraction environment at various pH values and the lipophilic characteristics of the compounds [49]. A disease correlation analysis of the identified compounds was performed based on the BATMAN-TCM online analysis tool. We examined which disease phenotypes CLDENs might be used to treat based on the disease targets information of identified compounds. The results indicated that CLDENs might have therapeutic potential for multiple severe combined immunodeficiency diseases and human immunodeficiency virus (Additional file 1: Table S3). These results demonstrated the possibility of applying CLDENs to immune system diseases.

Then, we performed differential proteomic analysis of C. roseus leaves and CLDENs. 1843 proteins were identified and quantified in the CLDENs group (Fig. 4C), compared to 3241 in the PLANT group (Additional file 1: Fig. S5). In CLDENs group, 86.04% of the identified proteins had molecular weights distributed between 10 and 80 kDa. After comparing the PLANT group and CLDENs group, a total of 1184 differentially expressed proteins were identified, of which 471 were significantly up-regulated and 713 were significantly down-regulated (Fig. 4D and E). Both up-regulated proteins and down-regulated proteins showed a clear tendency to localize to the chloroplast (Up: 28.45%, Down: 47.83%) and cytoplasm (Up: 27.18%, Down: 29.03%). But the up-regulated proteins had more proteins localized to the plasma membrane (Up: 19.11%, Down: 1.96%) (Fig. 4F and G).

Analysis around various up-regulated proteins revealed that CLDENs had an enrichment of transport and signaling proteins such as ABC transporter family members, enzymes like pyruvate decarboxylase, and endosomal membrane proteins represented by DnaJ homolog subfamily C GRV2-like protein. The ratio of relative quantification of significantly up-regulated proteins in C. roseus leaves and CLDENs were examined. The CLDENs/PLANT Ratio values of the top six up-regulated proteins were all greater than 100 (Additional file 1: Table S4), indicating that they were significantly enriched in CLDENs and can be thought of as the marker proteins of CLDENs. They were pyruvate decarboxylase, ABC transporter C family member 10, probable metal-nicotianamine transporter YSL6 isoform X1, glycerophosphodiester phosphodiesterase, DnaJ homolog subfamily C GRV2-like, and ABC transporter C family member 4-like.

Among these proteins significantly enriched in CLDENs (Additional file 1: Table S4), we found proteins associated with multivesicular and intraluminal vesicles, such as DnaJ homolog subfamily C GRV2-like protein, AP-3 complex subunit delta [50], Flotillin-like protein [51]. Particularly the DnaJ homolog subfamily C GRV2-like protein, a crucial protein in Arabidopsis thaliana's late endosome development. It is localized in the endosomal membrane and involved in multivesicular mediated vesicle trafficking [52]. Exosomes are thought to be tiny vesicles that are released outside the cell after binding to the plasma membrane by MVB [53]. The enrichment of these proteins provided evidence of CLDENs’ origin from MVB.

Immunostimulatory effects of CLDENs via TNF-α/ NF- κ B/PU.1 axis

Subsequently, we examined whether CLDENs played a role in inflammatory and immune processes. The secretion of cytokines could directly reflect the involvement of immune cells in the immune response [54]. It was found that CLDENs greatly stimulated the secretion of TNF-α (Fig. 5A). High levels of TNF-α were found in the RAW264.7 cells’ culture supernatant after 24 h of co-incubation with 120 g/ml CLDENs (3975.74 ± 469.24 pg/ml), which was over 30 times greater than the normal control group (135.96 ± 50.56 pg/ml) and exceeded the lipopolysaccharide (LPS) group (3650.09 ± 442.43 pg/ml). Besides, CLDENs dramatically increased the secretion of interleukin-6 (IL-6) by RAW264.7 cells and showed no discernible impact on interleukin-1β (IL-1β) or interleukin-10 (IL-10). In the concentration range of 0.98 ~ 62.5 μg/ml, CLDENs significantly stimulated RAW264.7 cells to secrete nitric oxide (Fig. 5B). From the protein level, an up-regulation of inducible nitric oxide synthase (iNOS) expression level was observed in RAW264.7 cells after CLDENs treatment (Fig. 5C). These results indicated that CLDENs promoted macrophages to secrete a variety of cytokines and activate the inflammatory response.

Fig. 5

Immunostimulatory effects of CLDENs in vitro. A CLDENs promoted the secretion of cytokines TNF-α and IL-6 from RAW264.7 cells, while it had no significant promotion effect on the secretion of IL-1β and IL-10. B CLDENs promoted nitric oxide release from RAW264.7 cells. C CLDENs increased the expression level of nitric oxide synthase in RAW264.7 cells. D, E Effect of CLDENs on the ability of RAW264.7 cells to phagocytose FITC-Dextran. Data were shown in mean fluorescence intensity (D) and histogram (E). F, G CLDENs enhanced RAW macrophage surface antigen CD86 and MHC II expression but had no discernible impact on CD206 expression. Data were shown in mean fluorescence intensity (G) and histogram (F). H CLDENs increased the mRNA transcription levels of M1-type related genes in RAW264.7 cells. I CLDENs reduced the mRNA transcription levels of M2-type related genes in RAW264.7 cells. J CLDENs activated the transduction of the NF-κB signaling pathway. K CLDENs up-regulated the expression level of PU.1 protein in RAW264.7 cells. L CLDENs up-regulated the mRNA transcript level of PU.1 gene in RAW264.7 cells. M Effect of CLDENs on the cell viability of primary spleen lymphocytes. N CLDENs promoted the secretion of IL-2 from primary splenic lymphocytes. Data were mean ± SD, n = 3; *P < 0.05, **P < 0.01 and ***P < 0.001 vs. Control

In addition, in order to detect the relationship between the structural integrity and activity of CLDENs, they were treated with enzymes, acids, bases, and surfactants before expose to RAW264.7 cells. The results showed that neither the acid–base treatment nor the 30-min enzymatic digestion treatment affected CLDENs’ ability to stimulate nitric oxide release from RAW264.7 cells. Similar to the previous results, this effect could be completely eliminated by surfactant and heat treatment (Additional file 1: Fig. S6). The above results indicating that structural integrity of CLDENs was essential for its immunomodulatory activity.

A FITC-Dextran fluorescent particle uptake experiment was used to detect the phagocytic function of macrophages. The results showed that CLDENs significantly enhanced the phagocytic function of RAW264.7 cells (Fig. 5D). The mean fluorescence intensity in 120 μg/ml CLDENs group was three times higher than that in the control group (Fig. 5E).

Macrophages usually differentiate into two major phenotypes M1 and M2 after being activated, which indicates the progression of the inflammatory response. Positive expression of macrophage surface antigen CD86 and MHC II is usually considered as M1-type macrophage, while CD206 is considered as M2-type macrophage [55, 56]. Flow cytometry was used to detect the influence of CLDENs on the expression of macrophage surface antigens on RAW264.7 cells. The results showed that CLDENs significantly increased the expression of CD86 and MHC II (Fig. 5F and G) though CD206 was unaffected by CLDENs. After 24 h of incubation with 120 μg/ml CLDENs, compared to the normal control group, RAW264.7 cells had significantly higher levels of mRNA transcription for M1 macrophage-related genes CD86, iNOS, IL-6, and TNF-α (Fig. 5H), but significantly lower levels for M2 macrophage-related genes Arginase-1 and CD206 (Fig. 5I). CLDENs greatly promoted the differentiation of RAW264.7 cells and induced their polarization toward the M1 type.

The above data demonstrated that CLDENs strongly stimulated macrophages to secrete TNF-α, enhanced phagocytosis of macrophages as well as promoted immune response-related cell differentiation. TNF-α can activate inflammation-related signaling pathways represented by the NF-κB pathway and induces downstream immunomodulation-related cascade responses [57]. Among which PU.1 is a key transcription factor in the regulation of immune cell growth, development, and function [58, 59]. Therefore, western blot assay was performed to detect the protein express level of NF-κB and PU.1. As expected, when RAW264.7 cells were treated CLDENs for 24 h, the expression and phosphorylation of NF-κB were increased (Fig. 5J, LPS as positive control). Also, CLDENs significantly increased the expression of PU.1 in RAW264.7 cells, both at the protein level (Fig. 5K, LPS as positive control) and at the transcriptional level (Fig. 5L). The above data demonstrated that CLDENs strongly stimulated macrophages to secrete TNF-α, enhanced phagocytosis of macrophages as well as promoted immune response-related cell differentiation.

Comparable effects were observed in splenic lymphocytes. Spleen lymphocytes were separated from mouse spleens and co-incubated with CLDENs for 24 h. CLDENs significantly promoted spleen lymphocyte proliferation (Fig. 5M). ELISA experiment results showed that CLDENs substantially improve splenic cells' release of interleukin-2 (IL-2) (Fig. 5N).

In short, CLDENs promoted the proliferation and differentiation of immune cells and facilitated their functional activation in vitro. This immunomodulatory effect was mediated through the TNF-α/NF-κB/PU.1 axis. Additionally, the intact vesicle structure and suitable temperature were required for CLDENs to function.

CLDENs alleviated cyclophosphamide-induced immunosuppression

An immunosuppressed mouse model was established by continuous 3-day administration of 80 mg/kg cyclophosphamide. In order to observe the possible toxic effects of CLDENs, we set the dose at a concentration gradient of nearly 3 times, that was 6 mg/kg, 20 mg/kg and 60 mg/kg. According to reports, the number of blood cell populations in peripheral blood can be used clinically to determine whether a patient is experiencing myelosuppression [60]. The routine blood tests showed that cyclophosphamide significantly reduced the number of white blood cells, lymphocytes, granulocytes, and monocytes in the peripheral blood of mice. Medium-dose and high-dose CLDENs significantly reversed the inhibitory effect of cyclophosphamide on blood cell counts (Fig. 6A-D). As for the low-dose group, CLDENs significantly alleviated the decline of white blood cells, lymphocytes, but no statistical difference was observed in granulocytes and monocytes (Fig. 6C and D).

Fig. 6

CLDENs alleviated cyclophosphamide-induced immunosuppression. A–D Effect of CLDENs on the number of white blood cells (A), lymphocytes (B), neutrophils (C), and monocytes (D) in the peripheral blood of immunosuppressed mice (n = 5). E, F Effect of CLDENs on the secretion levels of cytokines TNF-α (E) and IL-2 (F) in the serum of mice in a cyclophosphamide immunosuppression model (n = 3). G, H Effect of CLDENs on splenic lymphocyte subpopulations of immunosuppressed mice, number (G) and proportion (H). I Impact of CLDENs on the immunosuppressed mice's ability to remove carbon particles (n = 3) J Effects of CLDENs on cell cycle distribution of bone marrow cells in immunosuppressed mice (n = 3). K Effect of CLDENs on the relative body weight gain of cyclophosphamide-induced immunosuppressed mice (n = 10). L Effects of CLDENs on the liver and kidney tissues of cyclophosphamide-induced immunosuppression mice were analyzed by hematoxylin–eosin staining. Data were mean ± SD; *P < 0.05, **P < 0.01 and ***P < 0.001 vs. Blank; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. Model

Subsequently, to evaluate whether CLDENs still induced the secretion of inflammation-associated cytokines in vivo, the levels of the relevant cytokines in mouse serum were detected by ELISA assay. Very low levels of TNF-α were observed in the model group, and cyclophosphamide also inhibited the secretion of IL-2. Even in the presence of cyclophosphamide, CLDENs significantly increased serum TNF-α levels in vivo and exhibited a concentration dependence (Fig. 6E). Similarly, CLDENs significantly increased the level of IL-2 (Fig. 6F).

The CD4+/CD8+ ratio in mature T lymphocytes is considered to be a key indicator of the level of immunity in the body [61]. Mature T lymphocytes were isolated from the spleens of animals (CD3+). Cyclophosphamide significantly suppressed the number of CD3+CD4+ T cells (Fig. 6G) and decreased the ratio of CD4+/CD8+ T cells (Fig. 6H). Low-dose and medium-dose of CLDENs did not show a restorative effect on the absolute number of CD4+ T cells, but significantly increased the CD4+/CD8+ T cell ratio. In the high-dose group of CLDENs, both the number of CD4+ T cells and the ratio of CD4+/CD8+ T cells were significantly restored in the animals. These results suggested that CLDENs promoted positive regulation of immune function by improving the ratio of lymphocyte subpopulations.

Normal mouse Kupffer cells and spleen macrophages can remove foreign substances from the bloodstream. Blood was obtained at the 2nd and 10th minutes after the mice were given 25% Indian ink (a suspension of carbon particles) via tail vein, and the concentration of carbon particles in the blood was calculated. The function of macrophages in animals might be judged by calculating the clearance index K and phagocytosis coefficient α according to the speed at which carbon particles are cleared from the bloodstream. Experiments on carbon clearance revealed that cyclophosphamide dramatically decreased the clearance index K and phagocytosis coefficient α of mice (Fig. 6I and Table 1), which suggested that cyclophosphamide reduced the ability of mice to remove carbon particles from their blood circulation. After the administration of CLDENs (20 mg/kg and 60 mg/kg), the phagocytosis coefficient of cyclophosphamide immunosuppressed mice was dramatically increased, indicating that CLDENs had a positive protective effect on macrophage function. However, the animals in the low-dose group did not show a significant improvement in phagocytosis coefficient α.

Table 1 Effect of CLDENs on the carbon clearance experiment of cyclophosphamide-induced immunosuppression mice

The strong cell cycle arrest effect of cyclophosphamide is one of the inducements of its immunosuppression effect [17]. The distribution of the cell cycle was then examined using flow cytometry on bone marrow cells that had been extracted from mouse femurs. The findings demonstrated that CLDENs treatment alleviated the effect of cyclophosphamide-induced G1 arrest in mouse bone marrow cells (Fig. 6J).

It is reported that the transcription factor PU.1 determined HSC’s differentiation fate of macrophages, neutrophils,