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. 2017 Oct 16;9(10):2069-2082.
doi: 10.18632/aging.101302.

Natural Product Celastrol Suppressed Macrophage M1 Polarization Against Inflammation in Diet-Induced Obese Mice via Regulating Nrf2/HO-1, MAP Kinase and NF-κB Pathways

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Free PMC article

Natural Product Celastrol Suppressed Macrophage M1 Polarization Against Inflammation in Diet-Induced Obese Mice via Regulating Nrf2/HO-1, MAP Kinase and NF-κB Pathways

Dan Luo et al. Aging (Albany NY). .
Free PMC article

Abstract

Macrophage polarization is implicated in the inflammation in obesity. The aim of the present study was to examine the anti-inflammatory activities of botanical triterpene celastrol against diet-induced obesity. We treated diet-induced obese C57BL/6N male mice with celastrol (5, 7.5 mg/kg/d) for 3 weeks, and investigated macrophage M1/M2 polarization in adipose and hepatic tissues. Celastrol reduced fat accumulation and ameliorated glucose tolerance and insulin sensitivity. Celastrol down-regulated the mRNA levels of macrophage M1 biomarkers (e.g., IL-6, IL-1β, TNF-α, iNOS) in cell culture and in mice. The underlying mechanisms were investigated in murine macrophage RAW264.7 cells. Our results demonstrated that celastrol might control macrophage polarization through modulating the cross-talk between the following three mechanisms: 1) suppressing LPS-induced activation of MAP kinases (e.g., ERK1/2, p38, JNK) in a concentration dependent manner; 2) attenuating LPS-induced nuclear translocation of NF-κB p65 subunit in a time dependent manner; 3) activating Nrf2 and subsequently inducing HO-1 expression. HO-1 inhibitor SnPP diminished the inhibitory effects of celastrol on the activation of NF-κB pathway and the pro-inflammatory M1 macrophage polarization. Taken together, celastrol exhibited anti-obesity effects via suppressing pro-inflammatory M1 macrophage polarization. Thus, our results provide new evidence for the potential of celastrol in the treatment of obesity.

Keywords: adipose tissue; celastrol; inflammation; liver; macrophage polarization; obesity.

Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

Figures

Figure 1
Figure 1. Celastrol attenuated diet-induced obesity in C57BL/6N mice
(A) Celastrol promoted weight loss. Diet-induced obese mice were treated with celastrol (0, 5, 7.5 mg/kg/d) for 21 days, whereas control mice were fed with normal diet. Body weight was daily measured. (B) NMR determination of fat contents. After 21-day treatment, fat contents in mice were analyzed by Bruker minispec NMR analyzer. (C) Glucose tolerance test (GTT). After glucose injection, blood was collected and analyzed for glucose level. The area under curve (AUC) for each group was calculated. (D) Insulin tolerance test (ITT). After insulin injection, blood was collected and analyzed for glucose level. The AUC for each group was calculated. (E) Plasma level of IL-6. After 21-day treatment, plasma was isolated from mouse blood and measured by commercial mouse IL-6 ELISA kit. N=3; HFD, HFD only; C5, celastrol (5 mg/kg/d); C7.5, celastrol (7.5 mg/kg/d). (F) Plasma level of IL-1β. After 21-day treatment, plasma was isolated from mouse blood and measured by commercial mouse IL-1β ELISA kit. N=3; HFD, HFD only; C5, celastrol (5 mg/kg/d); C7.5, celastrol (7.5 mg/kg/d); **, p < 0.01; ***, p < 0.001
Figure 2
Figure 2. Celastrol differentially affected the expression of macrophage M1 and M2 biomarkers in epididymal adipose tissues and liver
(A) Detection of iNOS, COX-2, and arginase-1 in epididymal adipose tissues. After 21-day treatment, epididymal fat pads were recovered from mice, and stained with specific antibodies. CD68 was stained as pan-macrophage bomarker whereas the cell nuclei were stained with DAPI. The sections were imaged under a Zeiss LSM 780 confocal microscopy. Representative images were shown. Scale bar, 50 μm. (B) qRT-PCR quantification of macrophage M1 and M2 biomarkers in epididymal adipose tissues. Total RNAs were extracted from adipose tissues and analyzed by qRT-PCR technique using QuantiTect SYBR Green PCR Kit and specific DNA primers from Qiagen. N = 3; HFD, HFD only; C7.5, celastrol (7.5 mg/kg/d). (C) Detection of iNOS, COX-2, and arginase-1 in liver tissues. Livers were recovered from mice, and stained with specific antibodies. CD68 was stained as pan-macrophage biomarker whereas the cell nuclei were stained with DAPI. The sections were imaged under a Zeiss LSM 780 confocal microscopy. Representative images were shown. Scale bar, 50 μm. (D) qRT-PCR quantification of macrophage M1 and M2 biomarkers in liver tissues. Total RNAs were extracted from livers and analyzed by qRT-PCR technique using QuantiTect SYBR Green PCR Kit and specific DNA primers from Qiagen company. N = 3; HFD, HFD only; C5, celastrol (5 mg/kg/d); C7.5, celastrol (7.5 mg/kg/d); *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 3
Figure 3. Effects of celastrol on the expression of macrophage M1 and M2 biomarkers in RAW264.7 cells
(A) Cytotoxicity of celastrol. Following 48 h treatment, RAW264.7 cells were determined for the cell viability by a colorimetric MTT assay relative to the untreated controls (n = 3). **, p < 0.01 (Celastrol vs Control). (B) Effects of celastrol on the expression of iNOS, COX‐2 and arginase‐1 in LPS‐stimulated RAW264.7 cells. After 24 h treatment with LPS and/or celastrol, the cellular proteins were analyzed by Western blotting with specific antibodies and GAPDH (as loading control). The blots (n = 3) were quantified by a densitometric method. Representative blots were shown. (C) qRT‐PCR quantification of macrophage M1 and M2 biomarkers in RAW264.7 cells. After treatment with LPS and/or celastrol, the mRNA levels of specific biomarkers (n = 3) were analyzed by qRT‐PCR technique using Qiagen primers. C0.25, celastrol (0.25 μM); C0.5, celastrol (0.5 μM); C0.75, celastrol (0.75 μM); *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 4
Figure 4. Effects of celastrol on the activation of MAP kinases, NF-κB and Nrf2/HO-1 pathways in RAW264.7 cells
(A) Western blot analysis of MAP kinase activation in LPS-stimulated RAW264.7 cells. After treatment with LPS alone or in combination with celastrol, the cellular proteins were analyzed by Western blotting with specific antibodies. Representative blots were shown. The blots (n = 3) were quantified by a densitometric method. C0.25, celastrol (0.25 μM); C0.5, celastrol (0.5 μM); C0.75, celastrol (0.75 μM); **, p < 0.01; ***, p < 0.001. (B) Nuclear translocation of NF-κB p65 subunit. After treatment with 1 μg/ml LPS alone or in combination with 1 μM celastrol, the nuclear and cytoplasmic proteins were prepared and analyzed by Western blotting with specific antibodies. Lamin B and GAPDH were used as loading control. Representative blots were shown. (C) Nuclear translocation of Nrf2. After treatment with celastrol, the nuclear and cytoplasmic proteins were prepared and analyzed by Western blotting with specific antibodies. Lamin B and GAPDH were used as loading control. Representative blots were shown. (D) Induction of HO-1 expression. After 24 h treatment with celastrol, the cellular proteins were analyzed by Western blotting with specific antibodies and GAPDH (as loading control). Representative blots were shown. The blots (n = 3) were quantified by a densitometric method. *, p < 0.05; ***, p < 0.001.
Figure 5
Figure 5. HO-1 inhibitor SnPP attenuated NF-κB activation and macrophage biomarker expression against celastrol in LPS-stimulated RAW264.7 cells
(A) Restoration of NF-κB nuclear translocation against celastrol in LPS-stimulated macrophages. After drug treatment, the cells were stained for NF-κB p65 subunit, whereas the cell nuclei were stained with DAPI. The cells were imaged under a Zeiss fluorescence microscopy. Representative images were shown. Cela, celastrol (0.75 μM). Scale bar, 20 μm. (B) Specific up-regulation of iNOS expression against celastrol in LPS-stimulated macrophages. After 24 h treatment, the cellular proteins were analyzed by Western blotting with specific antibodies and GAPDH (as loading control). The blots (n = 3) were quantified by a densitometric method. Representative blots were shown. ***, p < 0.001. (C) Attenuation of celastrol inhibition on macrophage M1 and M2 biomarkers in LPS-stimulated RAW264.7 cells. After drug treatment, the expression of selected macrophage M1 and M2 biomarkers was quantified by qRT-PCR technique using Qiagen primers. C0.75, celastrol (0.75 μM); *, p < 0.05; **, p < 0.01; ***, p < 0.001. (D) Schematic illustration of the potential mechanisms. Celastrol activates Nrf2/HO-1 pathway while inhibits MAP kinases (e.g., ERK1/2, p38, JNK) and NF-κB pathway.

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