Celastrol exerts anti‐inflammatory effect in liver fibrosis via activation of AMPK‐SIRT3 signalling

Abstract Celastrol, a pentacyclic tritepene extracted from Tripterygium Wilfordi plant, showing potent liver protection effects on several liver‐related diseases. However, the anti‐inflammatory potential of celastrol in liver fibrosis and the detailed mechanisms remain uncovered. This study was to investigate the anti‐inflammatory effect of celastrol in liver fibrosis and to further reveal mechanisms of celastrol‐induced anti‐inflammatory effects with a focus on AMPK‐SIRT3 signalling. Celastrol showed potent ameliorative effects on liver fibrosis both in activated hepatic stellate cells (HSCs) and in fibrotic liver. Celastrol remarkably suppressed inflammation in vivo and inhibited the secretion of inflammatory factors in vitro. Interestingly, celastrol increased SIRT3 promoter activity and SIRT3 expression both in fibrotic liver and in activated HSCs. Furthermore, SIRT3 silencing evidently ameliorated the anti‐inflammatory potential of celastrol. Besides, we found that celastrol could increase the AMPK phosphorylation. Further investigation showed that SIRT3 siRNA decreased SIRT3 expression but had no obvious effect on phosphorylation of AMPK. In addition, inhibition of AMPK by employing compound C (an AMPK inhibitor) or AMPK1α siRNA significantly suppressed SIRT3 expression, suggesting that AMPK was an up‐stream protein of SIRT3 in liver fibrosis. We further found that depletion of AMPK significantly attenuated the inhibitory effect of celastrol on inflammation. Collectively, celastrol attenuated liver fibrosis mainly through inhibition of inflammation by activating AMPK‐SIRT3 signalling, which makes celastrol be a potential candidate compound in treating or protecting against liver fibrosis.


| INTRODUC TI ON
Compelling evidence linked inflammation to the development of liver fibrosis. 1,2 Currently, few effective drugs can be used to treat liver fibrosis, and suppressing liver inflammation is an effective strategy to control liver fibrosis. 3 It is well known that hepatic stellate cells (HSCs) activation is regarded as a critical step in mediating liver fibrosis, 4 and however, continuous secretion of inflammatory cytokines in liver tissue will lead to the persistent activation of HSCs.
Moreover, the activated HSCs itself also secrete inflammatory cytokines that further promote liver fibrosis. 5,6 Thus, suppressing inflammation and improving the inflammatory microenvironment are crucial for treatment of liver fibrosis.
The active ingredients extracted from Traditional Chinese Medicine have become important choices for the treatment of several chronic diseases, including liver fibrosis. 5,7 Celastrol is a pentacyclic triterpenoid compound 8,9 (the chemical structure of celastrol is shown in Figure 1), which is isolated from a common clinical used Traditional Chinese Medicine named Tripterygium wilfordii Hook F.
Celastrol possesses multiple pharmacological effects and showed potent anti-inflammatory effect against many disease models, [10][11][12][13] and however, few reports can be seen regarding the anti-inflammatory potential of celastrol in liver fibrosis. SIR2 is a family of histone deacetylases and is widely distributed in cells with multiple functions. 14 A total of seven members (SIRT1-SIRT7) have been identified in mammalian. 15 SIRT1 is predominantly nuclear and could modify the activity of target proteins through deacetylation, thus contributing to oxidative response and cell cycle control. 16 SIRT2 is the only sirtuin that is mainly located in the cytoplasm and plays roles in neurological disease, cancers and other diseases. 17 SIRT3, SIRT4 and SIRT5 are found in the mitochondrial, of which SIRT3 is closely associated with oxidative stress and has been demonstrated involved in many liver-related diseases. 18,19 SIRT6 is located in the nucleus with unique and important functions in maintaining cellular homoeostasis. 20 SIRT7 locates in nucleus and participated in the ribosomal RNA transcription, cell metabolism, cell stress and DNA damage repair. 21 AMPK has been proved involved in the pathophysiology of liver fibrosis, 22 and up-regulation of AMPK phosphorylation facilitated to the attenuation of liver fibrosis. 23 Interestingly, SIRT3 has been reported as a downstream effector of AMPK in several disease models, and activation of AMPK-SIRT3 signalling contributes to the improvement of mitochondrial function, thus alleviating the progress of diseases. 24,25 However, in liver fibrosis, whether celastrol regulate AMPK-SIRT3 signalling remains poorly understood. Moreover, whether activation of AMPK-SIRT3 signalling contributes to the anti-inflammatory effect of celastrol remains to be determined. In the current study, the effects of celastrol on liver fibrosis were investigated in vivo and in vitro, and the potential role of AMPK-SIRT3 signalling in liver fibrosis was assessed for the first time to reveal the underlying mechanisms. Primary antibody against PGC-1α was purchased from Affinity (AF5395). Hydroxyproline examination kit (A030-2-1) was purchased from Nanjing Jiancheng Bioengineering Institute. ELISA kits including IL-6 (H007), IL-18 (H015), IL-1β (H002), TNF-α (H052), IFN-γ (H025) and IL-10 (H009) were purchased from Nanjing Jiancheng Bioengineering Institute. Dorsomorphin (Compound C, an AMPK inhibitor) (S7840) was purchased from Selleck. The primers used in real-time PCR were from GenScript Co. Ltd. MegaTran 1.0 transfection reagent was from OriGene. SIRT3 enzyme activity detection kit (JK50288.2) was purchased from Shanghai Baoman Biotechnology Co., Ltd.

| Cell isolation, culture and transfection
Primary rat HSCs were isolated from male Sprague-Dawley rats weighing 180-220 g (Shanghai Slac Laboratory Animal) as described previously. 26 Isolated HSCs were cultured in DMEM with 10% foetal bovine serum and 1% antibiotics, and maintained at 37°C in a humidified incubator of 5% CO 2 and 95% air. Cell morphology was assessed using an inverted microscope with a Leica Qwin System (Leica). HSCs at passages 2-4 were used in experiments. SIRT3 siRNA, AMPK1α siRNA and matched negative control siRNA were purchased from RayBiotech. Cell transfection was performed using MegaTran 1.0 transfection reagent. The detail protocol was F I G U R E 1 Chemical structure of celastrol according to previously reported. The transfection efficiency was confirmed by Western blot analysis.

| Animals and experimental procedures
All experimental procedures were approved by the institutional and local committee on the care and use of animals of Nantong and blood was collected from common carotid arteries by using arterial intubation method in rats and then isolated the livers to calculate liver/body weight ratio. Meanwhile, a small piece of liver was cut and fixed by using 10% formalin to conduct histopathological and immunohistochemical studies. The rest liver was cut into pieces and frozen with liquid nitrogen rapidly to extract total RNA and hepatic proteins.

| Liver histopathology
Harvested liver tissues were fixed in 10% neutral buffered formalin and embedded in paraffin. Liver slices of 5 μm thick were prepared and stained with haematoxylin and eosin and masson's trichrome stain by using standard methods. For sirius red collagen staining, thin sections were deparaffinized and stained with picrosirius red for 1 hour at room temperature. After washes, sections on the slides were dehydrated in 100% ethanol and in xylene, and then they were mounted in Permount. Photographs were taken in a blinded fashion at random fields. Representative views of liver sections were shown.

| Biochemical analyses
Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) in serum samples were evaluated using enzyme-linked immunosorbent assay methods according to the kit protocols (Nanjing Jiancheng Bioengineering Institute). Experiments were performed in triplicate.

| Real-time PCR
Total RNA was extracted from rat liver samples or rat HSCs using Trizol reagent (Biouniquer Technology Co., Ltd.) and then subjected to reverse transcription to cDNA using the kits provided by TaKaRa Biotechnology Co., Ltd. according to the protocol.
Amplification kit was purchased from Bio-Rad Laboratories. GAPDH was used as the invariant control. Results were from triplicate experiments. The following primers of genes (Keygen) were used:

| Western blot analyses
RIPA buffer (Beyotime) supplemented with PMSF (Beyotime) was used form protein extraction from liver tissues and HSCs cells.
Protein concentration was measured using an Enhanced BCA Protein Assay kit (Beyotime). The protocol of Western blot analysis was according to previously reported. 28 Representative blots were from three independent experiments.

| Luciferase reporter assay
Hepatic stellate cells cells were cotransfected with SIRT3 promoter luciferase fusion plasmid and pRL-TK reporter plasmid (control reporter) using Lipofectamine 3000 reagent (Invitrogen). 14 Twenty-four hours later, cells were stimulated with PDGF-BB (20 ng/mL) for another 24 hours following treatment with celastrol (20 μmol/L) for 4 hours. Then, the fluorescence intensity was determined using a dual luciferase reporter assay system (Promega, Madison).

| SIRT3 enzyme activity assay
SIRT3 enzyme activity in rat's liver was detected using a SIRT3 enzyme activity detection kit (Baomanbio), using colorimetric method according to the protocol, and results were from triplicate experiments.

| Statistical analysis
All experimental data were presented as mean ± SD, and results were analysed by using Statistical Package for Social Sciences (SPSS) (IBM Incorporation) version 16.0 software. The significance of difference was determined by one-way ANOVA with the post hoc Dunnett's test. Values of P < .05 were considered statistically significant.

| Celastrol suppressed inflammation in rat fibrotic liver
Inflammation is directly linked to liver fibrosis, 1 and suppressing inflammation can effectively control liver fibrosis. We herein studied the effects of celastrol on the levels of inflammatory

| Celastrol attenuated liver fibrosis and inflammation in activated HSCs in vitro
As blocking, the HSCs activation helps to the control of liver fibrosis. 31

| Celastrol promoted SIRT3 expression and activity in activated HSCs and rat fibrotic livers
As sirtuin family participated in regulation of liver fibrosis, 32 Figure 7D) and in rat fibrotic livers ( Figure 7E). Altogether, celastrol increased SIRT3 expression in vivo and in vitro.

| Depletion of SIRT3 ameliorated the antiinflammatory effect of celastrol in activated HSCs
As celastrol increased SIRT3 expression, then, we wonder whether SIRT3 enhancement was essential for the inflammation inhibition

| Activation of AMPK signalling is essential for induction of SIRT3 enhancement by celastrol in activated HSCs
As celastrol increased SIRT3 expression in activated HSCs, however, how celastrol increase SIRT3 expression remains to be determined.

| Inhibition AMPK signalling decreased the antiinflammatory ability of celastrol in activated HSCs
The above data showed that celastrol could activate AMPK signalling in liver fibrosis, and thus, whether activation of AMPK signalling is essential for the inhibition of inflammation of celastrol in liver fibrosis was further clarified. As expected, celastrol remarkably suppressed the secretion of IL-6, IL-18 and IL-1β compared with those of the untreated group ( Figure 10A-C). Treatment with Compound C effectively attenuated the anti-inflammatory effect of celastrol ( Figure 10A-C).
The same results were obtained by treatment with AMPK1α siRNA ( Figure 10D-F). Altogether, activation of AMPK signalling is required for the anti-inflammatory effect of celastrol in activated HSCs.

| D ISCUSS I ON
In this study, we proved that the anti-fibrotic effect of celastrol was attributed to its anti-inflammatory effect, and such anti-inflammatory Celastrol is an active ingredient of TwHF, which is clinically used to treat the immune diseases. 36 Currently, celastrol is documented as a potent agent for treating inflammatory disorders. Celastrol attenuated the inflammatory response of the spinal cord after spinal cord injury by reducing the release of TNF-α, IL-1β and IL-18 inflammatory factors, and increasing the release of IL-10 cytokines. 37 Besides, celastrol showed potent anti-inflammatory effect against diet-induced obesity mainly by regulating macrophage polarization. 38 In parallel with these existing studies, our results further showing that celastrol had strong anti-inflammatory effect in liver fibrosis both in vivo and in vitro, suggesting that inflammation intervention was an effective strategy helps to the control of liver fibrosis.
As SIRT3 mainly located in mitochondria and is closely associated with oxidative stress, thus, the existing research mainly focus on its role on oxidative stress. 39 inflammation. 43 The current study showed that SIRT3 expression was increased by celastrol treatment in liver fibrosis, showing that SIRT3 is essential for the liver protection effect of celastrol against liver fibrosis. Moreover, further study demonstrated that SIRT3 disruption suppressed the secretion and expressions of inflammatory cytokines and improved the inflammatory microenvironment in liver fibrosis, indicating that SIRT3 intervene attenuated the anti-inflammatory effect of celastrol. Another important point is that TGF-β is considered as the key promoting factor in liver fibrosis, 44 as celastrol could up-regulate SIRT3, whether there is a regulatory role between SIRT3 and TGF-β? Previous study showed that SIRT3 could deacetylate and activate glycogen synthase kinase 3β (GSK-3β) to positively regulate its activity, which phosphorylated β-catenin and Smad to block TGF-β1 signalling and suppress tissue fibrosis. 45 Therefore, in the future study, the effect of celastrol on GSK-3β, phosphorylation of β-catenin and Smad will be investigated to clarify the regulatory role between SIRT3 and TGF-β.
AMPK is involved in the development of many disease models including liver fibrosis and activation of AMPK inhibited NF-κB signalling to alleviate liver inflammation and fibrosis. 45 Liver fibrosis can also be ameliorated by AMPK phosphorylation and blockade of mTOR-dependent cascades. 46 Consistent with this study, the present study showed that celastrol evidently increased the expression of phosphorylation of AMPK in liver fibrosis and inhibited AMPK by using selective inhibitor Compound C attenuated the anti-inflammatory effect of celastrol, suggesting that activation of AMPK might contribute to the anti-inflammatory effect of celastrol under liver fibrosis conditions. Another important point in our study was that PDGF-BB stimuli slightly increased the AMPK phosphorylation, but celastrol treatment significantly increased it, and we guess that it may be due to PDGF-BB stimuli HSCs formed a pathological environment (namely in vitro liver fibrosis model), and celastrol increased AMPK phosphorylation only in pathological environment but not in  remarkably had no obvious effect on AMPK phosphorylation. These data suggested that the interplay between AMPK and SIRT3 participated in the inflammatory inhibition effect of celastrol in liver fibrosis, and SIRT3 was a critical down-stream target of AMPK in liver fibrosis. In summary, we confirm that celastrol activated AMPK-SIRT3 signalling, and activation of AMPK-SIRT3 signalling facilitated to the anti-inflammatory effect of celastrol against liver fibrosis.

ACK N OWLED G EM ENTS
This work was supported by the grants from the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.