Berberine activates Sirt1 and decreases inflammation

Chronic inflammation is a major contributing factor in the pathogenesis of many diseases. Natural product berberine (BBR) exhibits potent anti-inflammatory effect in vitro and in vivo, while the underlying mechanisms remain elusive. Sirt1, a NAD+-dependent protein deacetylase, was recently found to play an important role in modulating the development and progression of inflammation. Thus, we speculate that Sirt1 might mediate the inhibitory effect of BBR on inflammation.

In LPS-stimulated RAW264.7 macrophages, BBR treatment sig- nificantly downregulated the expression of proinflammatory cytokines such as MCP-1, IL-6 and TNF-α. Importantly, BBR potently reversed LPS-induced down-regulation of Sirt1. Consistently, the inhibitory effects of BBR on proinflammatory cytokines expression was largely abrogated by Sirt1 inhibition either by EX527, a Sirt1 inhibitor or Sirt1 siRNA.

Further mechanistic studies revealed that BBR-induced inhibition of NF-κB is Sirt1- dependent, as either pharmacologically or genetically inactivating Sirt1 enhanced the IκΒα degradation, IKK phosphorylation, NF-κB p65 acetylation and DNA-binding activity. Taken together, our results provide the first evidence that BBR potently suppressed inflammatory responses in macrophages through inhibition of NF-κB signaling via Sirt1-dependent mechanisms.

 

Berberine (BBR), a major form of isoquinoline alkaloid isolated from plants such as Cotex phellodendri, Hydrastis canadensis and Rhizoma coptidis, has been used in Chinese medicine more than hundreds of years, showing multiple pharmacological activities [10]. Several reports showed that BBR possesses anti-inflammatory activities in vitro and in vivo [11]. In cultured RAW264.7 and primary peritoneal mac- rophages, BBR potently inhibited LPS-induced expression of proin- flammatory genes including IL-6, iNOS, and COX-2, mediated via AMPK activation [12].

Another study showed that BBR has anti-inflammatory effects on 3 T3-L1 adipocytes, evidenced by the down-regulation of mRNA level of several inflammation markers [13]. In either db/db or diet-induced obese mice, BBR reduced serum levels of TNF-α and IL-6 and inhibited the activation of NF-κB pathway in the adipose tissue [14,15]. In acute coronary syndrome patients, treatment for 30 days with BBR resulted in a significant reduction of serum IL-6, MCP-1 and C-reactive protein [16].

Meanwhile, our recent study reported that BBR inhibited M1 macrophages polarization and activation in adipose tissue in high fat diet-induced obese mice [17]. Nevertheless, the exact me- chanisms underlying the anti-inflammatory properties of BBR is not fully understood, especially in macrophages.

In the present study, we investigated the anti-inflammatory effects of BBR on macrophages in vitro and its association with Sirt1, given increasing evidence that Sirt1 functions as a crucial regulator of in- flammatory processes. Our results showed that BBR decreased the ex- pression of proinflammatory cytokines and inhibited the activation of NF-κB pathway in a Sirt1-dependent manner. These discoveries may help to further unveil the mechanisms of BBR’s anti-inflammatory ac- tion.

2. Materials and methods

2.1. Reagents

Berberine(≥98% purity), DMSO, and LPS (Escherichia coli, serotype 0111:B4) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine RNAiMAX reagent and Trizol reagent were purchased from Invitrogen (Carlsbad, CA, USA). PrimerScript RT reagent Kit and SYBR Green PCR kit were purchased from Takara (Dalian, China). The antibodies against IκΒα, NF-κB p65 and acetyl-NF-κB p65 (Lys310) were purchased from Abcam (Cambridge, MA, USA). Monoclonal an- tibodies to Sirt1, IKKβ, phospho-IKKβ, and horseradish peroxidase conjugated anti-rabbit and anti-mouse antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). β-actin mouse mono- clonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). BCA protein assay kit and West Pico chemiluminescent substrate were purchased from PIERCE (Rockford, IL, USA). Unless otherwise specified, all other reagents were analytic grade.

2.2. Cell lines and culture

Murine RAW264.7 macrophages were obtained from Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cul- tured in high glucose DMEM (GIBCO, Invitrogen Life Technologies Corporation, NY, USA) supplemented with 10% newborn calf serum (GIBCO, Invitrogen Life Technologies Corporation, Auckland, New Zealand), 100 U/ml penicillin and 100 μg/ml streptomycin in a humi- dified atmosphere with 5% CO2 at 37 °C. In all experiments, cells were acclimated for 24 h and then pretreated with berberine, berberine + EX527 at different concentrations 4 h prior to LPS (100 ng/ml) sti- mulation.

2.3. Cell viability assay

MTT assay was used to evaluate the effect of BBR on cell viability. Briefly, the RAW264.7 cells were cultured in 96-well plate at a density of 5 × 103 cells per well. After 24 h, cells were treated with various concentrations of BBR (1-20 μM) for 4 h, followed in the presence or absence of LPS (100 ng/ml) for another 20 h. Subsequently, 10 μl of MTT reagent was added into each well and incubated for 4 h. After removing the supernatant, 150 μl DMSO was added to dissolve the

crystals. Then, the absorption values were measured at 570 nm on a microplate reader. The optical density in the control group cells (no BBR and LPS treatment) was considered 100% viability.

2.4. Cell transfection with siRNA

The small interfering RNA (siRNA) duplexes for Sirt1 were com- mercially synthesized by Genepharma (Shanghai, China). The sequence was 5′-CCGUCUCUGUGUCACAAAUTT-3′. On the day before transfec- tion, RAW264.7 cells were seeded into six-well plates and grown in 2.0 ml of DMEM supplemented with 10% FBS. Then, Sirt1 siRNA or scrambled siRNA were transfected into RAW264.7 cells with Lipofectamine RNAiMAX reagent according to the manufacturer’s in- structions. After 72 h growth, RAW264.7 cells were used for the further interventions. Meanwhile, the knockdown in Sirt1 expression was evaluated by Western blot analysis.

2.5. Quantitative real-time reverse transcriptase polymerase chain Reaction (qRT-PCR)

The mRNA expression of MCP-1, IL-6 and TNF-α were measured by qPCR, and β-actin was used as an internal control for mRNAs. Total RNA was isolated from RAW264.7 cells with Trizol reagent according to the manufacturer’s instructions and reverse transcribed into cDNA using the PrimerScript RT reagent Kit. PCR amplification was performed with a SYBR Green PCR kit using the Applied Biosystems 7500 Real-Time PCR System in triplicates for three times independently.

The following primer sequences were used: MCP-1 forward 5′-ACC GGGA GGTG GTGA GGGT C-3′, MCP-1 reverse 5′-TGA GCCT ACGG GATC TGAA AGAC G-3′, IL-6 forward 5′-TGT GCAA TGGC AATT CTGA T-3′, IL-6 reverse 5′-CT CTGA AGGA CTCT GGC TTTG-3′, TNF-α forward 5′- CAACGCC CTCC TGGC CAA CG-3′, TNF-α reverse 5′-TCG GGGC AGCC TTG TCCC TT-3′, β-actin forward 5′-CGTTGAC ATCC GTAA AGAC C-3′ and β-actin reverse 5′-AAC AGTC CGCCTAGA AGCA C-3′. Parameters of PCR were: 5min 95°C for one cycle, 15s 95°C, 1min 60°C for 40 cycles, and 5 min 72 °C for one cycle. Reaction specificity was con- trolled by post-amplification melting curve analyses and gel electro- phoresis of products. The expression fold-changes were analyzed by the 2 − ΔΔCt relative quantitative method.

2.6. Cytokine measurements

RAW264.7 macrophages were seeded at a density of 1.0 × 106 cells/ml in 6-well plates, followed by incubation with serum- free DMEM containing BBR, BBR + EX527 with or without LPS (100 ng/ml) for 16 h. The levels of MCP-1, IL-6 and TNF-a in the culture supernatant were quantified using commercially available mouse ELISA kits from eBioscience (San Diego, CA, USA), according to the manu- facturer’s instructions. All experiments were performed in triplicate.

2.7. Western blot analysis

For Western blot analysis, RAW264.7 cells were harvested and suspended in ice-cold lysis buffer (50 mM Tris, 50 mM KCl, 20 mM NaF, 1 mM Na3VO4, 10 mM EDTA, 1% NP-40, 1 mM PMSF, and 5 μg/ml leupeptin, pH 8.8). Lysates were sonicated on ice and incubated for 30 min before being centrifuged at 12,000 rpm for 30 min at 4 °C.·Protein concentration was determined using the BCA protein assay kit. Equal amounts of protein were separated by 12% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA).

Membranes were incubated with blocking buffer (5% w/v BSA in TBS containing 0.1% Tween-20) for 2 h at room temperature, and incubated with a primary antibody overnight at 4 °C. After washing with TBS containing 0.1% Tween-20, the membranes were incubated for 2 h at room temperature with HRP-linked secondary antibodies. The mem- branes were then detected by the ECL plus Western blotting detection system (GE healthcare, Piscataway, NJ, USA) and quantified by the Quantity ONE software (Bio-Rad, Hercules, CA, USA). β-actin was used as an internal control.

2.8. NF-κB transcription activity assay

According to the manufacturer’s instructions, NF-kB p65 Transcription Factor Assay Kit was performed (Abcam, Cambridge, MA, USA) to detect NF-κB p65 DNA-binding activity. The absorbance at 450 nm was determined using a microplate reader.

2.9. Statistical analysis

Statistical analyses were performed using GraphPad PRISM 4.0. All values were expressed as mean ± standard errors of the means (SEMs). All data were from at least three independent experiments. Differences among multiple groups were determined by one-way ana- lysis of variance (ANOVA) followed by Tukey’s post-hoc test. Values were considered significantly different at P < 0.05.

3. Results

3.1. BBR suppressed proinflammatory cytokines expression in LPS- simulated RAW264.7 cells

In our study, we first examined the potential cytotoxicity of BBR by the MTT assay after incubating cells for 20 h in the absence or presence of LPS (100 g/ml). The results showed that BBR at the concentration up to 5 μM had no cellular toxicity on RAW264.7 cells. However, higher concentration of BBR showed obvious cytotoxicity (Fig. 1A). Thus, BBR concentrations ranging from 1 to 5 μM were selected for the subsequent experiments.

Since MCP-1, IL-6 and TNF-α have been reported to play critical roles during inflammatory responses [18], we then investigated the effects of BBR on these representative proinflammatory cytokines. As shown in Fig1.B-D, LPS treatment for 6 h significantly enhanced MCP-1, IL-6 and TNF-α mRNA levels in RAW264.7 macrophages. However, compared to the LPS-treated group, BBR (1, 2.5, 5 μM) pretreatment for 4 h dose-dependently suppressed MCP-1, IL-6 and TNF-α mRNA ex- pression. In addition, the effect of BBR on secretion of MCP-1, IL-6 and TNF-α was determined via ELISA assay. The results revealed that LPS

treatment for 16 h obviously up-regulated the levels of MCP-1, IL-6 and TNF-α. As expected, pretreatment with BBR also clearly inhibited the release of these cytokines in a dose-dependent manner (Fig. 2). Therefore, these findings showed that BBR significantly inhibited the LPS-induced expression of proinflammatory cytokines.

3.2. BBR enhanced the expression of Sirt1 in LPS-simulated RAW264.7 cells

There is accumulating evidence that Sirt1 exerts anti-inflammatory effects in various cells and its pharmacological activation may con- stitute a potential therapeutic strategy [19]. Thus, we sought to in- vestigate whether Sirt1 participated in LPS-induced inflammation re- sponses. RAW264.7 cells were exposed to LPS for 20 h and harvested. Total protein sample of cells was detected by Western blot analysis.

As shown in Fig. 3A and B, treatment with LPS produced marked decreases in Sirt1 protein levels. Interestingly, BBR (1, 2.5, 5 μM) pretreatment significantly reversed the LPS-induced down-regulation of Sirt1 in a dose-dependent manner. Based on these data, we postulate that the suppressive effects of BBR on LPS-induced inflammation involve Sirt1 up-regulation in RAW264.7 cells.

3.3. Blocking Sirt1 attenuated the protective effects of BBR on LPS- simulated RAW264.7 cells

To test the hypothesis, pharmacologically and genetically blocking experiments were conducted. First, RAW264.7 cells were pretreated with the specific Sirt1 antagonist Ex527 (10 μM) and or BBR (5 μM) for 4 h, and subsequently received LPS treatment for 6 h or 16 h. Consistent with the results shown in Fig.1and 2, BBR obviously down-regulated both the mRNA and protein levels of MCP-1, IL-6 and TNF-α.

However, this suppression was remarkably abrogated when the cells were pre- treated with Ex527 (Fig. 4). To provide direct evidence that Sirt1 is indeed relevant with the anti-inflammatory role of BBR, we specifically knocked down its expression with siRNA. Western blot analysis re- vealed that the Sirt1 level in the cells transfected with Sirt1 siRNA was 23.7% of that found in the cells transfected with scrambled siRNA (Fig. 3C and D). Down-regulation of Sirt1 also significantly reduced the suppressive effects of BBR on LPS-induced expression of proin- flammatory cytokines (Fig. 4). Above results together demonstrated that Sirt1 plays an important role in anti-inflammatory effects of BBR in

Fig. 1. Berberine (BBR) suppressed proinflammatory genes expression in LPS-stimulated RAW264.7 macrophages. (A): Effect of BBR on the viability of RAW264.7 cell. The cells were pretreated with BBR (1–20 μM) for 4 h, then stimu- lated with or without 100 ng/ml LPS for 20 h. Cell viability was determined via MTT assay and relative cell viability was calculated by compared with untreated control group. (B–D): Effect of BBR on the expression of proinflammatory genes. The cells were pretreated with BBR (1–5 μM) for 4 h before incubation with 100 ng/ml LPS for 6 h. Relative mRNA levels of each gene was analyzed with qRT-PCR and normalized to β-actin. Quantitative data were presented as mean ± SEM (n = 3). #p < 0.05 versus control, ##p < 0.01 versus control, ⁎p < 0.05, ⁎⁎p < 0.01 versus LPS alone.

Fig. 2. BBR prevented the LPS-stimulated release of MCP-1, IL-6 and TNF-α in RAW264.7 macrophages. The cells were pretreated with BBR (1–5 μM) for 4 h before incubation with 100 ng/ml LPS for 16 h. Then, secretion of MCP-1 (A), IL-6 (B) and TNF-α(C) in the media was evaluated by ELISA assay. Quantitative data were presented as mean ± SEM (n = 3). ##p < 0.01 versus control, ⁎p < 0.05 versus LPS, ⁎⁎p < 0.01 versus LPS.

Fig. 3. Effects of BBR on Sirt1 protein in LPS-stimulated RAW264.7 macrophages. (A-B): The cells were pretreated with BBR (1–5 μM) for 4 h before incubation with 100 ng/ ml LPS for 20 h. Then, cell lysates were prepared and analyzed for Sirt1 by Western blot analysis. β-actin was used as an internal control. (C–D): The cells were trans- fected with Sirt1 siRNA or control siRNA. Seventy-two hours after transfection, the expression of Sirt1 was mea- sured by Western blot analysis. β-actin was used as an in- ternal control. Quantitative data were presented as mean ± SEM (n = 3). ##p < 0.01 versus control.

LPS-stimulated RAW264.7 cells.

3.4. BBR inhibited NF-κB pathway activation in LPS-stimulated RAW264.7 cells

Given that NF-κB is a major transcriptional regulator for proin- flammatory cytokines, the effects of BBR on activation of NF-κB pathway were detected. As seen in Fig. 5A and B, after exposure to LPS for 30 min, IκΒα degradation was markedly caused. However, BBR (1, 2.5, 5 μM) pretreatment for 4 h dose-dependently inhibited LPS-in- duced IκΒα degradation. We further found that LPS-induced phos- phorylation of IKK in RAW 264.7 macrophages were significantly suppressed by treatment with BBR (Fig. 5C and D). Since acetylation of p65 at lysine 310 is required for full activation of NF-κB function [20], we examined the protein levels of its acetylation by Western blot

analysis. As shown in Fig. 5E and F, LPS treatment significantly strengthened acetylation of p65. However, the acetylation levels were attenuated to some degree by BBR treatment. Moreover, the NF-κB p65 DNA binding activity was measured in current study as well. As shown in Fig. 5G, LPS-enhanced the NF-κB p65 DNA binding activity were markedly blunted by BBR pretreatment. The results indicated that BBR broadly inhibited LPS-induced NF-κB pathway activation in RAW264.7 cells.

3.5. Blocking Sirt1 alleviated the inhibitory effect of BBR on NF-κB pathway in LPS-stimulated RAW264.7 cells

Sirt1/NF-κB axis is known to be involved in regulating expression of inflammatory cytokines [21]. To further elucidate the mechanism un- derlying the anti-inflammatory effects of BBR, we conducted a set of blocking experiments. The activation of NF-κB pathway was measured after blocking Sirt1. As shown in Fig. 6A–E, when Sirt1 was inhibited by EX527 or targeted siRNA, the suppressive effects of BBR on IκΒα de- gradation, IKK phosphorylation and NF-κB p65 acetylation were clearly reduced in LPS-simulated RAW264.7 cells. Blockage of Sirt1 also sig- nificantly attenuated the inhibitory effect of BBR on LPS-enhanced the NF-κB p65 DNA binding activity (Fig. 6G). These results from blocking experiments would hence, demonstrate that BBR-induced inhibition of NF-κB pathway was, at least in part, through activating Sirt1 in our system.

4. Discussion

Inflammation, as normal response of organisms to various injuries, plays a crucial role in host defense. However, uncontrolled and self- amplified inflammation is a major contributing factor in the patho- genesis of many diseases. Macrophages play a hub role in the initiation and modulation of host defense including inflammation, and can be activated by multiple proinflammatory stimuli such as LPS and TNF-α to trigger a cascade of inflammatory processes [2]. BBR is a major form of isoquinoline alkaloid isolated from plants such as Rhizoma coptidis, with promising anti-inflammatory effects [10].

However, the molecular mechanisms underlying these effects remain unclear. Thus, we used the well-characterized RAW264.7 macrophages to evaluate the inhibitory effect of BBR on LPS-induced inflammatory genes expression and to unravel the exact signaling pathway of action. In this study, we de- monstrated that BBR potently suppressed the proinflammatory re- sponses in macrophages. Furthermore, our data also revealed that this anti-inflammatory property of BBR was very likely resulted from the inactivation of NF-κB pathway through a Sirt1-dependent manner. To

the best of our knowledge, this is the first report demonstrating the anti- inflammatory effects of BBR and its association with Sirt1 in macro- phages.

It is well established that proinflammatory cytokines such as MCP-1, IL-6, IL-1β, COX2, and TNF-α play important roles in the pathogenesis of many diseases [22]. Some studies also showed that the expression of above cytokines were associated with the severity of inflammation and activation of macrophages both in vivo and in vitro [23].

Hence, blockage of these proinflammatory cytokines is becoming a promising strategy to alleviate and even cure some diseases. We previously de- monstrated that BBR administration improved insulin resistance and reduced serum levels of IL-6 and TNF-α in diet-induced obese mice [15]. Another study found that 5 μM BBR significantly reduced LPS- induced proinflammatory genes expression including IL-6, IL-1β, iNOS, MCP-1 and COX-2, suppressed the phosphorylation of MAPKs, and decreased the level of reactive oxygen species in macrophages [14].

In this study, the levels of MCP-1, IL-6, and TNF-α were used to indicate the severity of inflammation in LPS-stimulated RAW264.7 cells. As shown in Fig. 1, BBR pretreatment dose-dependently suppressed MCP- 1, IL-6 and TNF-α mRNA expression, compared to the LPS-treated group. Our present data confirmed that BBR is an anti-inflammatory substance. In addition, the effect of BBR on secretion of MCP-1, IL-6 and TNF-α was determined via ELISA assay. As expected, pretreatment with BBR also effectively inhibited the release of these cytokines in a dose- dependent manner. These results suggested that BBR exhibited in- hibitory effects on the production of these proinflammatory cytokines at both gene transcription and translation levels.

Sirt1 has been shown to be involved in the regulation of numerous development and homeostatic processes, including inflammatory re- sponse [4]. It cleaves the nicotinamide ribosyl bond of NAD+ and transfers the acetyl group from proteins to NAD+, thus leading to tighter chromosome structure and transcriptional repression of many inflammation-related genes [24].

Furthermore, some studies reported that BBR can activate Sirt1 in vivo and in vitro. Lin and colleagues demonstrated that BBR treatment for two weeks could protect against ischemia/reperfusion injury after orthotopic liver transplantation via activating Sirt1 [25]. Zhu et al. found that BBR exhibited a time-de- pendent effect on upregulation of Sirt1 in L02 cells, which could play an important role in its antioxidant activity under oxidative stress [26].

However, it is unclear whether BBR is also able to activate Sirt1 in macrophages. Our data showed that BBR (1, 2.5, 5 μM) pretreatment significantly reversed the LPS-induced down-regulation of Sirt1 in a dose-dependent manner. This implies that the inhibitory effect of BBR on inflammation in macrophages may be mediated by Sirt1. Attrac- tively, after inhibition of SRT1 with special antagonist EX527, the re- pressive effects of BBR on LPS-induced MCP-1, IL-6 and TNF-α ex- pression was remarkably abrogated. Knockdown of Sirt1 by siRNA also significantly reduced the suppressive effects of BBR on LPS-induced expression of proinflammatory cytokines.

Consistent with these finding, overexpression of Sirt1 or activation of Sirt1 by resveratrol have been shown to repress inflammatory responses in macrophage and pan- creatic β-cells [9,27]. Therefore, it seems that the anti-inflammatory effect of BBR on macrophages is indeed mediated by Sirt1. Currently, the signal pathways through which the Sirt1 levels decrease under in- flammatory conditions remain unclear. Further studies are required to

elucidate the exact mechanism of how BBR is able to activate Sirt1 in macrophages.

NF-κB pathway has a key function in the induction of major in- flammatory mediators. Under physiological conditions, NF-κB is held quiescent in the cytoplasm and binds to IκBα. When stimulated by LPS via Toll-like receptors, the IκBα is phosphorylated by IKK, which leads to ubiquitin-dependent proteasomal degradation and the subsequent translocation of NF-κB from cytoplasm to the nucleus [28]. Excitation of NF-κB results in transcription of a cascade of proinflammatory cyto- kines and chemokines, such as TNF-α, IL-1β, COX-2 and so on [29]. The expression of NF-κB-targeted genes is also modulated by acetylation of p65, a subunit of the heterodimetric proteins [30]. Mutational analysis showed that acetylation of p65 at lysine 310 is required for full acti- vation of NF-κB function [20].

Several studies found that the anti-in- flammatory property of BBR was resulted from the inactivation of NF- κB pathway [31,32]. And our previous study also discovered that BBR potently inhibited the activation of NF-κB pathway in adipose tissues of diet-induced obese mice [17]. Here in our study, BBR treatment sig- nificantly inhibited LPS-induced IκΒα degradation, IKK phosphoryla- tion, decreased the p65 acetylation and DNA-binding activity. Never- theless, the signaling pathway of BBR in mediation of NF-κB was not very clear.

Meanwhile, several studies showed that Sirt1 is able to ameliorate NF-κB-induced inflammatory responses through multiple mechanisms [21]. Sirt1 can directly combine with and deacetylates the p65 subunit at lysine 310 and subsequently inhibits NF-κB transcriptional activity [6,33]. Then, we further tested whether the actions of BBR were mediated by Sirt1 through the inhibition of NF-κB. We showed that addition of EX527 or knocking down Sirt1 by targeted siRNA significantly alleviated the inhibitory effect of BBR on NF-κB pathway in LPS-stimulated RAW264.7 cells. The results support our speculation that BBR-induced NF-κB inhibition was largely dependent on Sirt1 activation in macrophages.

5. Conclusions

Taken together, our results demonstrated that BBR potently sup- pressed inflammatory responses in macrophages through inhibition of NF-κB signaling via Sirt1-dependent mechanisms. These results may deepen our knowledge and understanding of BBR, and give some novel insight into its full application. However, further studies should be carried out to confirm this mechanism in vivo.