Nutr Res Pract.  2023 Jun;17(3):397-407. 10.4162/nrp.2023.17.3.397.

Role of heat shock protein 70 in regulation of anti-inflammatory response to curcumin in 3T3-L1 adipocytes

• Shin S
• Ajuwon KM

• KMID: 2542838
• DOI: http://doi.org/10.4162/nrp.2023.17.3.397

Abstract

BACKGROUND/OBJECTIVES
Curcumin is a well-known phytochemical with antiinflammatory effects. Heat shock protein (HSP) 70, an intracellular chaperone, inhibits proinflammatory signaling activation. Although curcumin has been shown to induce HSP70 expression in various cell types, whether HSP70 mediates the anti-inflammatory effects of curcumin in mature adipocytes remains unclear.
MATERIALS/METHODS
To assess the role of HSP70 in regulating the anti-inflammatory response to curcumin in adipocytes, fully differentiated 3T3-L1 adipocytes were treated with curcumin, lipopolysaccharide (LPS), and/or the HSP70 inhibitor pifithrin-μ (PFT-μ). The expression levels of HSP70 and proinflammatory cytokines were then measured.
RESULTS
Curcumin upregulated HSP70 expression at both protein and mRNA levels and attenuated LPS-induced Il6, Ptx3, and Ccl2> mRNA upregulation. PFT-μ tended to exacerbate the LPS-induced upregulation of Il6, Ptx3,Ccl2>, and Tnfa mRNA expression. However, on curcumin pretreatment, the tendency of PFT-μ to upregulate LPS-induced proinflammatory cytokine expression decreased or disappeared.
CONCLUSION
These results indicate that HSP70 is involved in the regulation of inflammatory responses but may not be crucial for the anti-inflammatory effects of curcumin in 3T3-L1 adipocytes.

Keyword

Curcumin; HSP70 heat-shock proteins; NF-kappa B; inflammation; adipocytes

Figure

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  Fig. 1 Effects of curcumin on heat shock protein levels. (A) HSP70; (B) HSP90. Fully differentiated 3T3-L1 adipocytes were treated with curcumin dissolved in dimethyl sulfoxide for 24 h. The protein levels were determined by Western blot with normalization relative to β-actin. Data are presented as means ± standard error of mean (n = 3).HSP, heat shock protein.a,bDifferent superscripts indicate significant differences at P < 0.05 by Duncan’s multiple comparison test. If a group has a common superscript with another group, it means they are not statistically different.

• 

  Fig. 2 Effects of curcumin and LPS on HSP70 and NF-κB signaling. (A) HSP70; (B) p-IκBα; (C) IκBα. Fully differentiated 3T3-L1 adipocytes were pretreated with 75 μM of curcumin dissolved in DMSO for 24 h and 10 μg/mL of LPS were treated for 30 min. The protein levels were determined by Western blot with normalization relative to β-actin. Data are presented as means ± standard error of mean (n = 6).LPS, lipopolysaccharide; HSP, heat shock protein; DMSO, dimethyl sulfoxide; Veh, vehicle; DV, dimethyl sulfoxide with vehicle; DL, dimethyl sulfoxide with lipopolysaccharide; CV, curcumin with vehicle; CL, curcumin with lipopolysaccharide.a,b,cDifferent superscripts indicate significant differences at P < 0.05 by Duncan’s multiple comparison test. If a group has a common superscript with another group, it means they are not statistically different.

• 

  Fig. 3 Effects of curcumin and LPS on pro-inflammatory cytokine expression. (A) Hspa1a; (B) Nfkb1; (C) Il6; (D) Ptx3; (E) Ccl2; (F) Tnfa. Fully differentiated 3T3-L1 adipocytes were pretreated with 75 μM of curcumin dissolved in DMSO for 24 h and 10 μg/mL of LPS were treated for 3 h. The mRNA levels were determined by quantitative real-time polymerase chain reaction with normalization relative to 18S rRNA. Data are presented as means ± standard error of mean (n = 3).LPS, lipopolysaccharide; DMSO, dimethyl sulfoxide; Veh, vehicle; Hspa1a, heat shock protein family A (heat shock protein 70) member 1A; Nfkb1, nuclear factor kappa B subunit 1; Il6, interleukin 6; Ptx3, pentraxin 3 (also known as tumor necrosis factor-inducible gene 14 protein); Ccl2, C-C motif chemokine ligand 2 (also known as monocyte chemoattractant protein-1); Tnfa, tumor necrosis factor α.a,b,c,dDifferent superscripts indicate significant differences at P < 0.05 by Duncan’s multiple comparison test.

• 

  Fig. 4 Effects of HSP70 inhibition on pro-inflammatory cytokine expression in 3T3-L1 cells pretreated with curcumin. (A) Hspa1a; (B) Nfkb1; (C) Il6; (D) Ptx3; (E) Ccl2; (F) Tnfa. Fully differentiated 3T3-L1 adipocytes were pretreated with 75 μM of curcumin dissolved in DMSO for 24 h, and 1 μM of PFT-μ for 24 h and 10 μg/mL of LPS for 3 h were treated. The mRNA levels were determined by quantitative real-time polymerase chain reaction with normalization relative to 18S rRNA. Data are presented as means ± standard error of mean (n = 3).HSP, heat shock protein; LPS, lipopolysaccharide; DMSO, dimethyl sulfoxide; Veh, vehicle; PFT-μ, pifithrin-μ; Hspa1a, heat shock protein family A (heat shock protein 70) member 1A; Nfkb1, nuclear factor kappa B subunit 1; Il6, interleukin 6; Ptx3, pentraxin 3 (also known as tumor necrosis factor-inducible gene 14 protein); Ccl2, C-C motif chemokine ligand 2 (also known as monocyte chemoattractant protein-1); Tnfa, tumor necrosis factor α.a,bDifferent superscripts indicate significant differences at P < 0.05 by Duncan’s multiple comparison test. If a group has a common superscript with another group, it means they are not statistically different.

• 

  Fig. 5 Effects of HSP70 inhibition on pro-inflammatory cytokine expression in 3T3-L1 cells pretreated with lower concentration of curcumin. (A) Hspa1a; (B) Nfkb1; (C) Il6; (D) Ptx3; (E) Ccl2; (F) Tnfa. Fully differentiated 3T3-L1 adipocytes were pretreated with 7.5 μM of curcumin dissolved in DMSO for 24 h, and 1 μM of PFT-μ for 24 h and 10 μg/mL of LPS for 3 h were treated. The mRNA levels were determined by quantitative real-time polymerase chain reaction with normalization relative to 18S rRNA. Data are presented as means ± standard error of mean (n = 3).HSP, heat shock protein; LPS, lipopolysaccharide; DMSO, dimethyl sulfoxide; Veh, vehicle; PFT-μ, pifithrin-μ; Hspa1a, heat shock protein family A (heat shock protein 70) member 1A; Nfkb1, nuclear factor kappa B subunit 1; Il6, interleukin 6; Ptx3, pentraxin 3 (also known as tumor necrosis factor-inducible gene 14 protein); Ccl2, C-C motif chemokine ligand 2 (also known as monocyte chemoattractant protein-1); Tnfa, tumor necrosis factor α.a,b,cDifferent superscripts indicate significant differences at P < 0.05 by Duncan’s multiple comparison test. If a group has a common superscript with another group, it means they are not statistically different.

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