β-Caryophyllene Acts as a Ferroptosis Inhibitor to Ameliorate Experimental Colitis

Abstract

Macrophage infiltration is one of the main pathological features of ulcerative colitis (UC) and ferroptosis is a type of nonapoptotic cell death, connecting oxidative stress and inflammation. However, whether ferroptosis occurs in the colon macrophages of UC mice and whether targeting macrophage ferroptosis is an effective approach for UC treatment remain unclear. The present study revealed that macrophage lipid peroxidation was observed in the colon of UC mice. Subsequently, we screened several main components of essential oil from Artemisia argyi and found that β-caryophyllene (BCP) had a good inhibitory effect on macrophage lipid peroxidation. Additionally, ferroptotic macrophages were found to increase the mRNA expression of tumor necrosis factor alpha (Tnf-α) and prostaglandin-endoperoxide synthase 2 (Ptgs2), while BCP can reverse the effects of inflammation activated by ferroptosis. Further molecular mechanism studies revealed that BCP activated the type 2 cannabinoid receptor (CB2R) to inhibit macrophage ferroptosis and its induced inflammatory response both in vivo and in vitro. Taken together, BCP potentially ameliorated experimental colitis inflammation by inhibiting macrophage ferroptosis. These results revealed that macrophage ferroptosis is a potential therapeutic target for UC and identified a novel mechanism of BCP in ameliorating experimental colitis.

Keywords: ferroptosis; inflammation; macrophage; type 2 cannabinoid receptor; ulcerative colitis; β-caryophyllene.

Figure 1

Ferroptosis is present in DSS-induced UC mice (n = 5). (A), Body weight and body weight changes in mice that received DSS treatment. (B), DAIs were determined according to scoring standards. (C), Colon length on the last day was measured and a histogram of colon length is shown. (D), H and E staining of colon tissues. Scale bar, 50 μm. (E), Levels of GSH, GPX activity and MDA in colonic tissues and serum iron levels of mice were examined using corresponding kits. (F), Protein expression levels of Acsl4, Fth1, Cox2 and Gpx4 were detected by western blotting. (G), Transmission electron microscopy of colon tissue mitochondria. Single white arrowheads indicate shrunken mitochondria; black arrowheads indicate increased mitochondrial membrane density. Scale bar, 1 μm. (H), Representative images of GPX4, MPO, COX2 and 4-HNE levels in colonic tissues of mice determined by immunohistochemistry staining. Scale bar, 50 μm. (I), Relative mRNA expression levels of Ptgs2, Acsl4, Slc7a11, Hamp, GPX4 and Fth1 were analyzed using RT-qPCR. Data are presented as the mean ± S.E.M. using Gapdh as a reference. * p < 0.05, ** p < 0.01, *** p < 0.001 versus mice from DSS group. 4-HNE, 4-hydroxynonenal; Acsl4, Acyl-CoA synthetase long-chain family member 4; Cox2, cyclooxygenase 2; DSS, dextran sulfate sodium; Fth1, ferritin heavy chain 1; GSH, glutathione; Gpx4, glutathione peroxidase 4; Hamp, hepcidin antimicrobial peptide; MDA, malondialdehyde; MPO, myeloperoxidase; Ptgs2, prostaglandin-endoperoxide synthase 2; Slc7a11, solute carrier family7 member 11.

Figure 2

Ferroptosis occurs in colon macrophages. (A), Representative flow cytometry images showing lipid peroxidation of colon macrophages of mice. (B), Representative images of F4/80 and 4-HNE levels in colonic tissues of mice were determined by colon tissue immunofluorescence staining, scale bar, 50 μm. 4-HNE, 4-hydroxynonenal; DAPI, 2−(4-Amidinophenyl)−6-indolecarbamidine dihydrochloride; DSS, dextran sulfate sodium.

Figure 3

Inflammation is induced in ferroptotic macrophages. RAW264.7 macrophages and BMDMs were incubated in medium containing RSL3 (500 nM), or RSL3 (500 nM) combined with Fer-1 (400 nM) for 6 h. (A,B), mRNA expression levels of Tnf-α and Ptgs2 in RAW264.7 macrophages and BMDMs were analyzed using RT-qPCR. Data are presented as the mean ± S.E.M. using Gapdh as a reference from three biologically independent samples. (C,D), Relevant protein levels of the MAPK signaling pathway and NF-κB pathways in RAW264.7 macrophages and BMDMs were analyzed by immunoblotting. β-Actin served as an internal reference. * p < 0.05, ** p < 0.01, versus RSL3 treatment group. BMDM, bone marrow-derived macrophage; Fer−1, ferrostatin−1; Ptgs2, prostaglandin-endoperoxide synthase 2; RSL3, ras-selective lethal small molecules 3; TNF-α, tumor necrosis factor α; Jnk, c-Jun N-terminal kinase; p-Jnk, phosphorylated-c-Jun N-terminal kinase; Erk1/2, extracellular regulated protein kinase1/2; p-Erk1/2, phosphorylated-extracellular regulated protein kinase1/2; Ikkα/β, inhibitor of kappa B kinase; p-Ikkα/β, phosphorylated-inhibitor of kappa B kinase; IκBα, inhibitor of nuclear factor kappa-B; p-IκBα, phosphorylated-inhibitor of nuclear factor kappa-B; P65, nuclear factor kappa-B; p-P65, phorylated-nuclear factor kappa-B.

Figure 4

BCP exerts an inhibitory effect on RSL3-induced macrophage ferroptosis. RAW264.7 macrophages or BMDMs were treated with 500 nM RSL3 in the presence of different concentrations of BCP or 400 nM Fer-1 for 6 h. (A–F), Representative flow cytometry images, percentage of lipid ROS and fluorescence intensity showing lipid peroxidation of RAW264.7 macrophages and BMDMs after different treatments. (G), Cell viability was measured using a CCK−8 assay. (H), Release of LDH in cell supernatants was measured using LDH assay kit. (I), GPX activity was determined using GPX activity kit. (J), The concentrations of MDA were determined using TBARS assay kit. Data are presented as the mean ± S.E.M. at least three biologically independent samples. n.s., not significant, # p < 0.05, ### p < 0.001 versus control group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus RSL3 treatment group. BCP, β-Caryophyllene; BMDM, bone marrow-derived macrophage; Fer−1, ferrostatin−1; GPX, glutathione peroxidase; LDH, lactate dehydrogenase; MDA, malondialdehyde; RSL3, ras-selective lethal small molecules 3.

Figure 5

BCP ameliorates mitochondrial damage induced by RSL3. RAW264.7 macrophages or BMDMs were treated with 500 nM RSL3 in the presence of different concentrations of BCP or 400 nM Fer−1 for 6 h. (A,B), Representative JC-1 staining images showing mitochondrial membrane potential changes of RAW264.7 macrophages and BMDMs. Scale bar, 200 μm. (C,D), Transmission electron microscopy of RAW264.7 macrophage and BMDM mitochondria after different treatments. Scale bar, 500 nm. BCP, β-Caryophyllene; BMDM, bone marrow-derived macrophage; Fer−1, ferrostatin−1; RSL3, ras-selective lethal small molecules 3.

Figure 6

BCP treatment inhibits ferroptosis-induced inflammation. RAW264.7 macrophages or BMDMs were treated with 500 nM RSL3 in the presence of different concentrations of BCP or 400 nM Fer−1 for 6 h. (A,B), Relative mRNA levels of Tnf-α and Ptgs2 in RAW264.7 macrophages and BMDMs were detected by RT-qPCR. Data are presented as the mean ± S.E.M. using Gapdh as a reference. ## p < 0.01, ### p < 0.001 versus control group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus RSL3 treatment group. (C,D), Relevant protein levels of the MAPK and NF-κB signaling pathway in RAW264.7 macrophages and BMDMs were analyzed by immunoblotting. β-Actin was used as an internal reference. All data are representative of or combined from at least three independent experiments. BCP, β-caryophyllene; BMDM, bone marrow-derived macrophage; Fer−1, ferrostatin−1; Ptgs2, prostaglandin-endoperoxide synthase 2; RSL3, ras-selective lethal small molecules 3; TNF-α, tumor necrosis factor α; Jnk, c-Jun N-terminal kinase; p-Jnk, phosphorylated-c-Jun N-terminal kinase; Erk1/2, extracellular regulated protein kinase1/2; p-Erk1/2, phosphorylated-extracellular regulated protein kinase1/2; Ikkα/β, inhibitor of kappa B kinase; p-Ikkα/β, phosphorylated-inhibitor of kappa B kinase; IκBα, inhibitor of nuclear factor kappa-B; p-IκBα, phosphorylated-inhibitor of nuclear factor kappa-B; P65, nuclear factor kappa-B; p-P65, phorylated-nuclear factor kappa-B.

Figure 7

BCP inhibits macrophage ferroptosis by activating the CB2R. RAW264.7 macrophages or BMDMs were treated with 500 nM RSL3 in the presence of BCP (20 μM), CB2R antagonist AM630 (5 μM) or both AM630 and BCP for 6 h. (A–F), Representative flow cytometry images, percentage of lipid ROS and fluorescence intensity showing lipid peroxidation of RAW264.7 macrophages and BMDMs after different treatments. (G), Cell viability of RAW264.7 cells and BMDMs was measured using CCK-8 assay kit. (H), Release of LDH in cell supernatants was measured using LDH assay kit. (I), GPX activity was determined using GPX activity kit. (J), The concentrations of MDA were determined using TBARS assay. Data are presented as the mean ± S.E.M. n.s., not significant, # p < 0.05, ## p < 0.01, ### p < 0.001 versus control group; * p < 0.05, ** p < 0.01, *** p < 0.001 versus RSL3 treatment group. All data are combined from at least three independent experiments. AM630, 6-Iodopravadoline; BCP, β-caryophyllene; BMDM, bone marrow-derived macrophage; Fer−1, ferrostatin−1; GPX, glutathione peroxidase; LDH, lactate dehydrogenase; MDA, malondialdehyde; RSL3, ras-selective lethal small molecules 3.

Figure 8

BCP ameliorates RSL3-induced mitochondrial damage via activation of the CB2R. RAW264.7 macrophages or BMDMs were treated with 500 nM RSL3 in the presence of BCP (20 μM), CB2R antagonist AM630 (5 μM) or both AM630 and BCP for 6 h. (A), Representative JC-1 staining images showing mitochondrial membrane potential changes of RAW264.7 macrophages and BMDMs. Scale bar, 200 μm. (B), Transmission electron microscopy of RAW264.7 macrophage and BMDM mitochondria after different treatments. Scale bar, 500 nm. AM630, 6-Iodopravadoline; BCP, β-caryophyllene; BMDM, bone marrow-derived macrophage; Fer-1, ferrostatin-1; RSL3, ras-selective lethal small molecules 3.

Figure 9

BCP regulates the expression of ferroptosis-related molecules by activating the CB2R. RAW264.7 macrophages or BMDMs were treated with 500 nM RSL3 in the presence of BCP (20 μM), CB2R antagonist AM630 (5 μM) or both AM630 and BCP for 6 h. (A), Relative mRNA levels of Gpx4 and Hamp in RAW264.7 macrophages and BMDMs were detected by RT-qPCR. Data are presented as mean ± S.E.M. using Gapdh as a reference. n.s., not significant, # p < 0.05, control group; * p < 0.05, versus RSL3 treatment group. (B) Relevant protein levels of ferroptosis in RAW264.7 macrophages and BMDMs were analyzed by immunoblotting. β-Actin was used as an internal reference. (C) All data are representative of or combined from at least three independent experiments. Acsl4, Acyl-CoA synthetase long-chain family member 4; Alox5, lipoxygenase 5; AM630, 6−Iodopravadoline; BCP, β-caryophyllene; BMDM, bone marrow-derived macrophage; Cox2, cyclooxygenase 2; Fth1, ferritin heavy chain 1; Gpx4, glutathione peroxidase 4; Hamp, hepcidin antimicrobial peptide; RSL3, ras-selective lethal small molecules 3.

Figure 10

BCP inhibits ferroptosis-induced expression of inflammatory genes via activation of CB2R. RAW264.7 macrophages or BMDMs were treated with 500 nM RSL3 in the presence of BCP (20 μM), CB2R antagonist AM630 (5 μM) or both AM630 and BCP for 6 h. (A,B), Relative mRNA levels of Tnf-α and Ptgs2 in RAW264.7 macrophages and BMDMs were detected by RT-qPCR. Data are presented as mean ± S.E.M. using Gapdh as a reference. n.s., not significant, # p < 0.05, control group; * p < 0.05, versus RSL3 treatment group. (C,D), Relevant protein levels of the MAPK signaling pathway and NF-κB pathways in RAW264.7 macrophages and BMDMs were analyzed by immunoblotting. β-Actin was used as an internal reference. All data are representative of or combined from at least three independent experiments. AM630, 6−Iodopravadoline; BCP, β-caryophyllene; BMDM, bone marrow-derived macrophage; Ptgs2, prostaglandin-endoperoxide synthase 2; RSL3, ras-selective lethal small molecules 3; TNF-α, tumor necrosis factor α; Jnk, c-Jun N-terminal kinase; p-Jnk, phosphorylated-c-Jun N-terminal kinase; Erk1/2, extracellular regulated protein kinase1/2; p-Erk1/2, phosphorylated-extracellular regulated protein kinase1/2; Ikkα/β, inhibitor of kappa B kinase; p-Ikkα/β, phosphorylated-inhibitor of kappa B kinase; IκBα, inhibitor of nuclear factor kappa-B; p-IκBα, phosphorylated-inhibitor of nuclear factor kappa-B; P65, nuclear factor kappa-B; p-P65, phorylated-nuclear factor kappa-B.

Figure 11

BCP attenuates ferroptosis induced by DSS by activating CB2R in vivo. Mice were given 3% DSS for 7 days and were treated with BCP (50 mg/kg, p.o.) alone, with the CB2R selective antagonist AM630 alone, or with AM630 (30 min before) plus BCP. (A–D), Content of GSH, GPX activity and MDA in colonic tissues and the levels of serum iron (n = 6) were examined using corresponding kits. (E), Relative mRNA levels of Ptgs2, Acsl4, Hamp, Gpx4 and Fth1 in colonic tissues of C57BL/6J mice were detected. Data are presented as the mean ± S.E.M. of 6 colonic tissue samples using Gapdh as a reference. (F), Acsl4, Cox2, Fth1 and Gpx4 protein expression in mice was determined by western blot analysis. β-Actin was used as an internal reference. n.s., not significant, # p < 0.05, ## p < 0.01, ### p < 0.001 compared with mice from control group; * p < 0.05, ** p < 0.01, *** p < 0.001 compared with mice from DSS group. Acsl4, Acyl −CoA synthetase long−chain family member 4; AM630, 6-Iodopravadoline; BCP, β-caryophyllene; Cox2, cyclooxygenase 2; DSS, dextran sulfate sodium; Fth1, ferritin heavy chain 1; GSH, glutathione; Gpx4, glutathione peroxidase 4; Hamp, hepcidin antimicrobial peptide; MDA, malondialdehyde; Ptgs2, prostaglandin-endoperoxide synthase 2.

Figure 12

BCP ameliorates macrophage ferroptosis via activation of the CB2R in vivo (n = 6 in each group). (A), Representative images of immunohistochemical staining of colonic tissues for 4-HNE of colonic tissues. Scale bar, 100 μm. (B), Representative images of transmission electron microscopy of colonic tissues after different treatments. Scale bar, 500 nm. (C), Representative images of double immunofluorescence staining of colonic tissues for F4/80 and 4-HNE. Scale bar, 50 μm. 4-HNE, 4-hydroxynonenal; AM630, 6-Iodopravadoline; BCP, β-caryophyllene; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride; DSS, dextran sulfate sodium.