Gut microbiota-palmitoleic acid-interleukin-5 axis orchestrates benzene-induced hematopoietic toxicity

Figures & data

Figure 1. Alterations in body weight, and phenotype of hematopoietic damage in mice after 45 days of benzene exposure.

(a) Body weight trend in benzene-exposed and control mice (n = 5/group); (b) Representative pathological images magnified 1×, 20× and 40× of H&E staining of mouse femur sections (n = 3/group); (c) Schematic diagram of HSCs renewal and differentiation; (d – g) Alteration of HSCs (LSK, MPPs, LT-HSCs, and ST-HSCs) in BM cells of control and benzene-exposed mice by flow analysis (n = 8/group); (h – k) Alterations in blood cell counts (WBC, RBC, HGB, and PLT) in the control and benzene-exposed mice (n = 5/group). *P < .05, **P < .01, compared to the control group.

Figure 2. Absolute quantification of cytokines in plasma.

(a) The levels of inflammatory cytokines in mouse plasma following 45-day benzene exposure (n = 5/group); (b) Dose-dependent expression of IL-5 and IL-13 in mouse plasma (n = 5/group); (c) Correlation of plasma levels of IL-5 and IL-13 with hematopoietic-related indices in mice (Spearman analysis); (d) Plasma levels of IL-5 and IL-13 in healthy controls and benzene-exposed workers (n = 162). *P < .05, **P < .01, ns: P > .05, compared to the control group.

Figure 3. Altered gut microbiota composition in mice after benzene exposure.

(a) Changes in the intestinal microbial profile of mice after 15, 30 and 45 days of benzene exposure; (b) LEfSe histogram of differential gut microbiota at 45 days of benzene exposure (LDA score > 4.0) (n = 5/group); (c) Gut microbiota with the greatest difference in relative abundance between the control and benzene-exposed groups were examined by 16S rDNA sequencing (n = 5/group) and qPCR quantification (n = 3/group); (d) Correlation analysis of differential gut microbiota and phenotype of hematopoietic damage. *P < .05, **P < .01, ***P < .001, compared to the control group.

Figure 4. Altered plasma metabolite profiles in mice after 45 days of benzene exposure.

(a) Heat map analysis of 79 identified plasma differential metabolites in control and benzene-exposed groups (n = 5/group); (b) Percentage distribution of differential metabolites; (c) Representative plasma LCFAs expression in the control and benzene-exposed groups (n = 5/group); (d) Associations of LCFAs with differential gut microbiota and hematopoietic damage parameters. *P < .05, **P < .01, compared to the control group.

Figure 5. Gut microbiota contribute to benzene-induced hematopoietic damage by regulating fatty acid metabolism.

(a) Schematic diagram of fecal microbiota transplant experiment; (b) Abundance of Lactobacillus murinus in the intestinal contents of mice after fecal microbiota transplant was determined by qPCR (n = 4/group); (c) Alteration of LCFAs levels in recipient mice (n = 6/group); (d – e) Changes in critical rate-limiting enzyme for fatty acid oxidation (n = 3/group); (f) Alteration of plasma IL-5 levels in recipient mice (n = 6/group); (g – o) Alterations in BM pathology, peripheral blood counts (n = 8/group) and HSCs (n = 6/group) in recipient mice. *P < .05, **P < .01, ns: P > .05, compared to the control group.

Figure 6. Elevated LCFAs promote benzene-induced hematopoietic toxicity.

(a) Schematic diagram of the fatty acid intervention experiment; (b) Effect of supplementation with MA and POA on body weight in mice (n = 6/group); (c-d) Plasma MA and POA levels in mice after 45 days of exposure (n = 4/group); (e-f) The protein expression levels of CPT2 were analyzed by IHC (×50 Magnification, Scale bar: 20 μm) (n = 5/group); (g) Alteration of plasma IL-5 in mice after LCFAs supplementation (n = 4/group); (h – k) Changes in the number of peripheral blood cells in mice after LCFAs supplementation (n = 6/group); (l – o) Changes in the proportion of HSCs in BM of mice after LCFAs supplementation (n = 3/group). *P < .05, **P < .01, ***P < .001, compared to the control group; ^#P < .05, ^##P < .01, compared to the benzene-exposure group.

Figure 7. Effect of CPT2 overexpression on benzene-induced hematopoietic damage.

(a) Schematic diagram of CPT2 overexpression experiment; (b) mRNA levels of CPT2 in mouse BM cells after injection of AAV-CPT2 (n = 4/group); (c-f) Protein levels of CPT2 in mouse BM cells were detected by western blotting assay and immunohistochemical assays (×50 Magnification, Scale bar: 20 μm) (n = 3/group); (g) CPT2 overexpression suppresses the abnormal elevation of plasma IL-5 in mice (n = 10/group); (h – k) CPT2 overexpression ameliorates benzene-induced abnormal HSCs ratios (n = 4/group); (l – o) CPT2 overexpression ameliorates benzene-induced peripheral blood cell decline (n = 10/group). *p < .05, **P < .01, ***P < .001, compared to the control+AAV-GFP group; ^#P < .05, ^##P < .01, compared to the benzene+AAV-GFP group.

Figure 8. Probiotic supplementation protects the mice from benzene-induced hematopoietic damage.

(a) Experimental design of oral probiotics; (b) Lactobacillus murinus colonization of mouse intestinal contents after oral supplementation of probiotics was analyzed by qPCR (n = 3/group); (c) POA levels in mouse plasma after oral administration of probiotics were detected by UPLC-MS/MS (n = 6/group); (d) IL-5 levels in mouse plasma were detected by ELISA after oral administration of probiotics (n = 6/group); (e) BM pathology in mice after oral administration of probiotics (×40 Magnification, Scale bar: 20 μm); (f – k) Changes in peripheral blood cell counts in mice after oral administration of probiotics (n = 8/group); (h – m) Changes in the proportion of BM HSCs in mice after oral administration of probiotics (n = 8/group). *P < .05, **P < .01, ns: P > .05.

Data availability statement

The microbial raw data reported in this paper have been deposited in the China National Center for Bioinformation (https://ngdc.cncb.ac.cn) and the accession numbers are PRJNA1068769.