Celastrol, an oral heat shock activator, ameliorates multiple animal disease models of cell death

Abstract

Protein homeostatic regulators have been shown to ameliorate single, loss-of-function protein diseases but not to treat broader animal disease models that may involve cell death. Diseases often trigger protein homeostatic instability that disrupts the delicate balance of normal cellular viability. Furthermore, protein homeostatic regulators have been delivered invasively and not with simple oral administration. Here, we report the potent homeostatic abilities of celastrol to promote cell survival, decrease inflammation, and maintain cellular homeostasis in three different disease models of apoptosis and inflammation involving hepatocytes and cardiomyocytes. We show that celastrol significantly recovers the left ventricular function and myocardial remodeling following models of acute myocardial infarction and doxorubicin-induced cardiomyopathy by diminishing infarct size, apoptosis, and inflammation. Celastrol prevents acute liver dysfunction and promotes hepatocyte survival after toxic doses of thioacetamide. Finally, we show that heat shock response (HSR) is necessary and sufficient for the recovery abilities of celastrol. Our observations may have dramatic clinical implications to ameliorate entire disease processes even after cellular injury initiation by using an orally delivered HSR activator.

Fig. 1

Celastrol ameliorated DOX-induced cardiomyopathy and protected against myocardial ischemia injury

Fig. 2

Celastrol treatment preserved cardiac function after myocardial infarction. a Echocardiography was performed before MI and after 7 days or after 28 days which showed a preservation of function with CEL treatment (**P < 0.01, *P < 0.05, two-way ANOVA followed by Bonferroni’s test). b A representative picture of Masson’s trichrome staining of heart sections (scale bar = 3.0 mm, magnification ×2.5). c Quantitative histological assessment of viable tissue at day 28 showed a reduced infarct expansion in CEL-treated mice as compared to controls (*P = 0.026, Mann–Whitney test). d CEL treatment suppressed CD68-positive cell infiltration in the myocardium at 3 days post-MI by histological evaluation (scale bar = 480 μm, magnification ×10). e Quantitative assessment showed a significant decrease in the number of CD68⁺ cells in MI hearts after CEL treatment as compared to MI hearts (*P = 0.034, Mann–Whitney test). f–h Quantitative analysis of inflammation-related cytokines and chemokines (*P < 0.05, **P < 0.01, *** P < 0.001, Kruskal–Wallis test followed by Dunn’s post hoc test). Data are represented as mean ± SEM

Fig. 3

Celastrol treatment enhanced survival after thioacetamide-induced liver toxicity in mice. a After TAA exposure, the CEL + TAA (n = 9, 87 %) group when compared to the TAA group (n = 8, 0 %, P < 0.0001, log-rank (Mantel–Cox) test) had a significant survival rate by Kaplan–Meier plot. b TAA exposure showed lobular disorganization and CV hemorrhaging which was absent with CEL treatment (PT portal triad, CV central vein, black arrowhead hemorrhaging and necrotic liver parenchyma, white arrowhead normal undamaged hepatocytes, scale bar = 480 μm, magnification ×10). c TUNEL apoptosis assays on liver sections obtained from untreated (control), TAA group, and TAA + CEL group. DAPI was used to counterstain nuclei. d. Quantitative analysis of TUNEL assay suggested that CEL treatment antagonized TAA exposure. TUNEL-positive nuclei are expressed as the average of five fields/sample (scale bar = 230 μm, magnification ×20). e The AST levels were significantly elevated by TAA exposure but brought to normal levels with CEL treatment. Data are analyzed by one-way ANOVA, followed by Kruskal–Wallis test, and presented as mean ± SEM

Fig. 4

Cell viability increased by CEL treatment against cytotoxins. a The cell viability of DOX-exposed H9c2 cells was significantly increased with CEL measured by MTT. b. The cell viability of TAA-exposed Huh7 cells was significantly increased with CEL. Data are analyzed by one-way ANOVA followed by Kruskal–Wallis test with Dunn’s comparisons. c Immunostaining of DOX-exposed H9c2 cells and TAA-exposed Huh7 cells in the absence (upper panel) or presence (lower panel) of CEL showed an inhibition of active caspase-3 expression (scale bar = 140 μm, magnification ×40). d Quantitative analysis of active caspase-3 expression showed a significant decrease in apoptotic cells with CEL (non-parametric t test followed by Mann–Whitney’s analysis). e FACS analysis of active caspase-3 expression in H9c2 cells exposed to DOX and treated with CEL. f FACS analysis of active caspase-3 expression in and Huh7 cells exposed to TAA. After drug exposure by cytometric histogram plot (left) and quantitative analysis (right) showed a higher expression level of active caspase-3 in DOX- and TAA-exposed cells when compared in DOX + CEL or TAA + CEL. FACS data were analyzed by non-parametric t test followed by Mann–Whitney’s analysis. g Immunostaining showed that APAP exposure resulted in almost complete cell death after 96 h, while in the presence of CEL, minimal cell death was observed (scale bar = 160 μm, magnification ×40). Data are represented as mean ± SEM)

Fig. 5

Celastrol increased HSPs and preserved essential transcription factors. a, b Immunoblot analysis showed an increase in active caspase-3 expression after exposure to DOX and TAA in H9c2 and Huh7 cells, respectively. GATA4 in H9c2 and Hnf-1α and Hnf-4α in Huh7 cells were decreased in a time-dependent manner after exposure to DOX or TAA. c, d GATA4 protein expression in H9c2 cells and Hnf-1α and Hnf-4α protein levels in Huh7 cells were restored to normal levels after CEL treatment by immunoblot analysis. Cleaved caspase-3 expression was significantly reduced. e CEL treatment increased hsp70 and hsp27 without showing any effect on hsp90 and hsp40 in a dose-dependent manner by immunoblot analysis. f CEL treatment restored GATA4 and hsp27 protein levels in mice hearts after DOX exposure analyzed by immunoblot analysis

Fig. 6

The HSR is necessary for the cytoprotective action by celastrol. a Mouse embryonic fibroblasts (MEFs) were cultured with DOX in the presence or absence of CEL. Increasing CEL doses inhibited DOX toxicity measured by MTT assay. b CEL treatment could not inhibit DOX toxicity in HSF1^−/− MEFs. c, d Immunoblot analysis showed that CEL treatment increased hsp70 and hsp27 without affecting hsp90 and hsp40 in HSF1^+/+ MEFS but not HSF^−/− MEFs. e Knockdown of HSF1 by siRNA, drug administration, and sample collection were performed as outlined. f Immunoblot analysis of H9c2 cells with non-targeting siRNA showed that the depletion in GATA4 protein levels caused by DOX administration was restored to normal levels after treatment with CEL, while CEL failed to restore GATA4 protein levels in H9c2 cells targeted with HSF1 siRNA. g Quantification of GATA4 protein levels after mock siRNA and HSF1 siRNA transfection in H9c2 cells. h H9c2 cells were treated with triptolide (TTD) in the presence or absence of CEL treatment or DOX exposure as indicated for 18 h. Immunoblot analysis showed that the inhibition of HSP70 by TTD correlated with inhibition of GATA4 protein. i Survival rates were higher in the CEL + DOX group (n = 9, 78 %) when compared to the DOX group (n = 10, 25 %, P = 0.0002, log-rank (Mantel–Cox) test) as shown by the Kaplan–Meier plot. TTD blocked the increased survival rate seen with CEL (CEL + TTD + DOX, n = 9, 22 %). No mortality was observed in the CNT and TTD group