s2fieldsofstudy: Biology Medicine

text:
Kallistatin treatment attenuates lethality and organ injury in mouse models of established sepsis

Introduction Kallistatin levels in the circulation are reduced in patients with sepsis and liver disease. Transgenic mice expressing kallistatin are resistant to lipopolysaccharide (LPS)-induced mortality. Here, we investigated the effect of kallistatin on survival and organ damage in mouse models of established sepsis. Methods Mice were rendered septic by cecal ligation and puncture (CLP), or endotoxemic by LPS injection. Recombinant human kallistatin was administered intravenously six hours after CLP, or intraperitoneally four hours after LPS challenge. The effect of kallistatin treatment on organ damage was examined one day after sepsis initiation, and mouse survival was monitored for four to six days. Results Human kallistatin was detected in mouse serum of kallistatin-treated mice. Kallistatin significantly reduced CLP-induced renal injury as well as blood urea nitrogen, serum creatinine, interleukin-6 (IL-6), and high mobility group box-1 (HMGB1) levels. In the lung, kallistatin decreased malondialdehyde levels and HMGB1 and toll-like receptor-4 (TLR4) synthesis, but increased suppressor of cytokine signaling-3 (SOCS3) expression. Moreover, kallistatin attenuated liver injury, serum alanine transaminase (ALT) levels and hepatic tumor necrosis factor-α (TNF-α) synthesis. Furthermore, delayed kallistatin administration improved survival in CLP mice by 38%, and LPS-treated mice by 42%. In LPS-induced endotoxemic mice, kallistatin attenuated kidney damage in association with reduced serum creatinine, IL-6 and HMGB1 levels, and increased renal SOCS3 expression. Kallistatin also decreased liver injury in conjunction with diminished serum ALT levels and hepatic TNF-α and TLR4 expression. In cultured macrophages, kallistatin through its active site increased SOCS3 expression, but this effect was blocked by inhibitors of tyrosine kinase, protein kinase C and extracellular signal-regulated kinase (ERK), indicating that kallistatin stimulates a tyrosine-kinase-protein kinase C-ERK signaling pathway. Conclusions This is the first study to demonstrate that delayed human kallistatin administration is effective in attenuating multi-organ injury, inflammation and mortality in mouse models of polymicrobial infection and endotoxemia. Thus, kallistatin therapy may provide a promising approach for the treatment of sepsis in humans.

Introduction
Sepsis is a systemic inflammatory response caused by microbial infection or bacterial products, such as lipopolysaccharide (LPS) [1]. Despite many years of dedicated research, sepsis remains one of the most frequent causes of mortality in critically ill patients, and contributes to significant economic costs in the United States [1][2][3]. Because numerous signaling cascades are triggered during sepsis, selective blocking of inflammatory mediators is not sufficient to arrest this process [3]. Inflammation and multi-organ dysfunction are closely associated with sepsis-induced lethality [4]. Sepsis is mediated by early (tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6)) and late (high mobility group box-1 (HMGB1)) inflammatory cytokines in response to infection [1]. Although the underlying pathophysiology of sepsis has not been completely elucidated, TNF-α and HMGB1 upregulation is known to play a crucial role in the systemic inflammatory response [5][6][7]. Moreover, suppressor of cytokine signaling-3 (SOCS3), a feedback inhibitor of LPS-induced inflammation and immune response [8], has also been shown to be a key player in inhibiting NF-κB-mediated pro-inflammatory cytokine production and HMGB1 release [9,10]. LPS stimulates SOCS3 expression in macrophages, possibly through activation of protein kinase C (PKC), phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK1/2) [11,12]. Furthermore, SOCS3 deficiency was found to promote inflammation in macrophages, whereas SOCS3 gene delivery reduced the mortality of mice with LPSinduced endotoxic shock [9,13]. Thus, approaches that broadly target the inhibition of systemic inflammation could provide effective strategies for the treatment of sepsis.
Kallistatin was first purified and characterized from human plasma as a novel serine proteinase inhibitor (serpin) and a specific tissue kallikrein inhibitor [14]. Kallistatin is an acidic glycoprotein with a pI of 4.6 and a molecular weight of 58 kDa [14,15]. As an endogenous protein, kallistatin exhibits pleiotropic effects in inhibiting inflammation, apoptosis and oxidative stress in animal models and cultured cells [16][17][18][19][20][21]. The normal plasma level of kallistatin in healthy subjects, measured by a specific ELISA, is 22.1 ± 3.5 μg/ml [22]. Circulating kallistatin levels are reduced in patients with septic syndrome and liver disease, as well as in mice with LPS-induced endotoxemia [22,23]. Transgenic mice expressing rat kallistatin display prolonged survival when subjected to endotoxic shock [23]. Moreover, kallistatin SERPINA4 gene transfer attenuates mortality, inflammation, and liver and skin damage in mice with Gram-positive streptococcal infection [24]. Kallistatin competes with TNF-α binding to cultured endothelial cells through its heparin-binding domain, thereby antagonizing TNF-α-induced NF-κB activation, pro-inflammatory gene expression and subsequent inflammatory response [21]. Likewise, kallistatin's heparin-binding site is essential for blocking HMGB1induced inflammatory gene expression in endothelial cells [25]. Therefore, kallistatin is capable of inhibiting the inflammatory responses of both early (TNF-α) and late (HMGB1) cytokines. We recently reported that kallistatin pre-treatment attenuated kidney injury, inflammatory gene expression and mortality in cecal ligation and puncture (CLP)-induced septic mice [25]. In this study, we further investigated the effect and potential mechanism of delayed kallistatin administration after onset of sepsis on mortality and organ injury in mouse models of polymicrobial sepsis and endotoxic shock.

Purification and characterization of recombinant human kallistatin
Recombinant human kallistatin was secreted into the serum-free medium of cultured human embryonic kidney cells (HEK293T), and culture medium was concentrated by ammonium sulfate precipitation followed by nickelaffinity and heparin-affinity chromatography as previously described [25,26]. Human recombinant wild-type kallistatin, heparin-binding site mutant kallistatin and active site mutant kallistatin were purified from Escherichia coli as previously described [26].
Cecal ligation and puncture-induced sepsis and survival study CD-1 mice (male, seven to eight-weeks-old; Harlan, Indianapolis, IN, USA) were housed in a germ-free environment. All procedures complied with the standards for care and use of animal subjects as stated in the National Research Council's Guide for the Care and Use of Laboratory Animals. The protocol for all animal studies was approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina (approval number: ARC#3158). All surgery was performed under anesthesia to minimize suffering. CLP was performed as described previously [25]. Briefly, the cecum was ligated at the colon juncture and then punctured twice with a 21-gauge needle. All animals were then fluid-resuscitated subcutaneously with 0.8 ml normal sterile saline. The sham operation was performed in the same way as CLP, but without ligation and puncture of the cecum. Mice were randomly assigned to one of four groups (n = 5 per group): 1) sham group receiving phosphate-buffered saline (PBS); 2) CLP control group receiving PBS; 3) CLP + KS3 group receiving 3 mg/kg body weight of kallistatin; or 4) CLP + KS10 group receiving 10 mg/kg body weight of kallistatin. Mice were injected intravenously with 0.1 ml PBS or recombinant human kallistatin in 0.1 ml PBS six hours after surgery. Mice (Harlan, Indianapolis, IN, USA) were killed 24 hours after CLP, and serum and tissues were collected for biochemical and histological analyses. For the survival study, mice were randomly assigned to one of three groups (n = 16 per group): 1) sham group receiving PBS; 2) CLP control group receiving PBS; or 3) CLP + KS20 group receiving 20 mg/kg body weight of kallistatin. At six hours after CLP surgery, mice were injected intravenously with 0.1 ml PBS or recombinant kallistatin in 0.1 ml PBS. Mouse survival was monitored every 24 hours for a total of six days.

Lipopolysaccharide-induced endotoxemia and survival study
Endotoxemia was induced in CD-1 mice (male, seven to eight-weeks-old) by intraperitoneal injection of LPS (15 mg/kg body weight; E. coli LPS 0111:B4; Sigma-Aldrich, St. Louis, USA) dissolved in sterile saline. Mice were randomly assigned to one of four groups (n = 6 per group): 1) control group receiving sterile saline; 2) LPS control group receiving sterile saline; 3) LPS + KS10 group receiving 10 mg/kg body weight of kallistatin; or 4) LPS + KS20 group receiving 20 mg/kg body weight of kallistatin. At four hours after LPS injection, mice were injected intraperitoneally with 0.1 ml of sterile saline or recombinant kallistatin in 0.1 ml sterile saline. Mice were sacrificed at 22 hours after LPS challenge, and serum and tissues were collected for biochemical and histological analyses. For the survival study, mice were assigned to one of two groups (n = 16 per group): 1) LPS control group receiving sterile saline; or 2) LPS + KS20 group receiving 20 mg/kg body weight of kallistatin. Mice were injected intraperitoneally with 0.1 ml sterile saline or recombinant kallistatin in 0.1 ml sterile saline four hours after LPS injection. Mouse survival was monitored every 12 hours for a total of four days.
Measurements of kallistatin, blood urea nitrogen, creatinine, TNF-α, IL-6, HMGB1, malondialdehyde and alanine transaminase levels in serum Human kallistatin levels in the serum were determined by enzyme-linked immunosorbent assay (ELISA) specific for human kallistatin [22]. Blood urea nitrogen (BUN) and serum creatinine were measured with QuantiChrom Urea and Creatinine Assay Kits (BioAssay Systems, Hayward, CA, USA), respectively. Serum TNF-α levels were determined by a mouse TNF-α ELISA kit (EMD Millipore, Billerica, MA, USA). IL-6 levels in serum were measured using a mouse IL-6 ELISA kit (eBioscience, San Diego, CA, USA). Serum HMGB1 levels were determined by a mouse HMGB1 ELISA kit (Antibodies-online Inc., Atlanta, GA, USA). The levels of lung malondialdehyde (MDA) were determined as previously described [27]. Serum alanine transaminase (ALT) levels were measured using an ALT colorimetric assay kit (Cayman Chemical Company, Michigan, MI, USA).

Tissue staining
For histological studies, kidney and liver were fixed with 4% paraformaldehyde, dehydrated, embedded and cut into 4-μm sections. Tissue sections were stained with hematoxylin and eosin (H&E) for examination of morphological damage.

Real-time reverse transcription-polymerase chain reaction
Total RNA was extracted from lung, kidney and liver tissues and cultured macrophages with TRIzol® reagent (Invitrogen, Carlsbad, CA, USA), following the manufacturer's protocol. cDNA was synthesized with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Quantitative real-time PCR was performed by Prism 7300 Real Time PCR System (Applied Biosystems) using TaqMan Gene Expression Master Mix (Applied Biosystems) in a final reaction volume of 20 μl with each primer. The following primers were purchased from Applied Biosystems: mouse GAPDH (Mm 99999915_gl), mouse HMGB1 (Mm 00849805_gl), mouse TLR4 (Mm 00445274_m1), mouse TNF-α (Mm 00443258_ml) and mouse SOCS3 (Mm 00545913_s1). A negative control without cDNA did not produce any amplicons. Data were analyzed with 2 −ΔΔCt value calculation, using GAPDH for normalization.

Western blot analysis
Macrophages were lysed with ice-cold RIPA lysis buffer (10 mM Tris, pH 7.4, 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin A, Sigma-Aldrich, St. Louis, USA). All lysed samples were kept on ice for 30 minutes, and centrifuged for 10 minutes at 4°C at 12,000 g. The supernatant was collected and stored at −20°C until further analysis. Cell lysates were subjected to 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membranes (GE Heathcare, Waukesha, WI, USA) were blocked with 7% milk in Tris-buffered saline-Tween 20 (TBST, 20 mM Tris, 500 mM NaCl, and 0.1% Tween 20) for one hour. After washing with TBST twice, membranes were incubated with primary antibody overnight at 4°C. The following primary antibodies were used: polyclonal anti-phospho-p44/42 MAPK (ERK1/2) and polyclonal anti-p44/42 MAPK (ERK1/2) (Cell Signaling, Boston, MA, USA). The membranes were washed twice with TBST and incubated with Peroxidase from horseradish (HRP) conjugated secondary antibody (Cell Signaling, Boston, MA, USA) in blocking buffer for one hour. After washing three times with TBST, immunoreactive bands were visualized by incubation with ECL plus detection reagents (GE Healthcare, Waukesha, WI, USA) for five minutes and exposure to BioMax light film (Thermo Scientific, Waltham, MA, USA). The densitometry of bands was quantified with ImageJ2 software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis
Data are expressed as means ± standard error of the mean (SE). Statistical significance was determined by analysis of variance (ANOVA) with Fisher's probable least-squares difference test or log-rank (Mantel-Cox) test, using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). A value of P <0.05 was considered statistically significant.

Delayed kallistatin administration prolongs survival of polymicrobial septic and endotoxemic mice
To determine the effect of kallistatin on CLP-induced mortality, kallistatin (20 mg/kg) was injected intravenously into mice at six hours after CLP surgery. A mortality study was conducted and the survival of CLP mice was monitored every 24 hours for a total of six days. Sham-operated mice (n = 16) exhibited 100% survival. All CLP control mice died within four days after CLP, whereas kallistatin administration (20 mg/kg) increased the survival rate by 38% compared to CLP control mice (n = 16, P <0.05; Figure 4A). To determine the effect of kallistatin on LPSinduced mortality, sterile saline or kallistatin (20 mg/kg) was injected intraperitoneally into mice four hours after LPS injection (15 mg/kg). Mouse survival was monitored every 12 hours for a total of four days. Treatment with kallistatin significantly improved the survival rate by 42% compared to the LPS control mice (n = 16, P <0.05; Figure 4B).

Discussion
This is the first study to demonstrate that kallistatin administration after onset of sepsis protects against lethality and multi-organ damage in mouse models of both polymicrobial infection and LPS-induced endotoxemia. Human kallistatin not only improved the survival of mice with established sepsis, but also significantly reduced organ injury in the kidney, lung and liver. Kallistatin treatment markedly lowered systemic inflammation as indicated by decreased circulating levels of the pro-inflammatory cytokines TNF-α, IL-6 and HMGB1. Our findings also indicate that kallistatin dramatically upregulated SOCS3 expression, a negative regulator of inflammation, in the lung and kidney. Moreover, kallistatin's active site plays a crucial role in inducing SOCS3 expression via activation of a tyrosine kinase-PKC-ERK pathway in cultured macrophages. Our previous studies indicated that kallistatin, through its heparin-binding site, blocks TNF-α and HMGB1-induced inflammatory responses [21,25]. Collectively, these findings reveal a protective role and unique mechanisms of kallistatin in mortality, systemic inflammation and multi-organ damage in mice with established sepsis. Importantly, as sepsis is the second leading cause of death in non-coronary ICU patients [30], kallistatin may be a novel strategy for combating sepsis syndrome in humans.
Severe sepsis is characterized by systemic inflammation, organ damage and a high rate of mortality. The crucial role of inflammatory cytokines in the pathogenesis of sepsis has been convincingly demonstrated [7]. Numerous cytokines and inflammatory mediators, including IL-1, IL-6, IL-12 and TNF-α, are documented to be released in the early phase of sepsis, while HMGB1 is released at the late phase [7]. High serum concentrations of TNF-α are correlated with sepsis-induced mortality [31]. It has also been shown that blockade of IL-6 and HMGB1 increases the survival of septic animals with established sepsis [32,33]. Our present study shows that delayed administration of human kallistatin markedly attenuated kidney, liver and lung injury in association with reduced serum levels of TNF-α, IL-6 and/or HMGB1 in mice with established sepsis. Kallistatin, through its heparin-binding site, inhibits TNF-α-and HMGB1induced inflammation by antagonizing their binding to endothelial cell surfaces [21,25]. Therefore, kallistatin can effectively lower both early and late phase systemic inflammatory cytokine levels and their downstream effects during sepsis. This suggests that the beneficial effect of kallistatin on sepsis-induced organ injury and mortality is mainly attributed to its inhibition of inflammatory cytokine expression. SOCS3 plays an important role in protection against sepsis as it is a negative regulator of LPS-induced inflammation [34]. A previous report showed that kallistatin protects against LPS-induced inflammation and TNF-α production via upregulation of macrophage SOCS3 expression [35]. Our current study also indicates that kallistatin attenuated inflammation and organ injury in association with increased SOCS3 expression in the lung and kidney of mice with polymicrobial infection or LPS-induced endotoxic shock. LPS and inflammatory cytokines were shown to induce SOCS3 expression via PKC-ERK or PI3K-ERK signaling [11]. Our results indicate that kallistatin increased SOCS3 expression in cultured macrophages via activation of a tyrosine kinase-PKC-ERK signaling pathway, as kallistatin's effects on ERK phosphorylation and SOCS3 expression were blocked by inhibitors of tyrosine kinase, PKC and ERK1/2. Kallistatin's effect on SOCS3 expression was not affected by LY294002 or L-NAME, indicating an event independent of the PI3K-Akt-NOS pathway (data not shown). Kallistatin contains two structural elements: an active site and a heparin-binding domain [28,29]. Kallistatin, via its heparin-binding site, interacts with cell surface heparan sulfate, thus blocking the binding of TNF-α or HMGB1 to their receptors and subsequent inflammatory responses [21,25]. In the present study, we showed that kallistatin, through its active site, stimulated SOCS3 expression by activating a cell surface tyrosine kinase, and thus PKC-ERK signaling. Therefore, kallistatin protects against sepsis-induced inflammation, organ damage and mortality by antagonizing TNF-α-and HMGB1-mediated inflammatory gene expression, and by inducing SOCS3 synthesis ( Figure 8). Kallistatin was shown to increase endothelial NOS expression and activation, and thus NO formation, leading to inhibition of vascular inflammation [20,36]. Taken together, these findings indicate that kallistatin is a unique anti-inflammatory agent by modulating differential signaling pathways.
LPS stimulates expression of SOCS3, which in turn negatively regulates LPS-induced TLR signaling [8,34]. TLRs elicit systemic inflammatory responses and contribute to organ injury and high mortality in animal models of sepsis [37]. TLR2 and TLR4 expression are increased in septic patients, and inhibition of TLR signaling and expression was shown to improve the survival of septic mice [38]. TLR4 is considered to be the main receptor mediating the inflammatory activity of HMGB1 [39]. Kallistatin pretreatment was found to reduce TLR4 expression in the lungs and kidneys of CLP mice [25]. The present study also shows that delayed kallistatin administration decreased sepsis-induced TLR4 expression in the lung and liver. Therefore, kallistatin most likely modulates TLR signaling and expression through induction of SOCS3 synthesis, leading to protection against sepsis-induced inflammatory response and organ injury.
Dysfunction of the kidney, lung and liver are common morbidities associated with sepsis [40]. Interestingly, the kallistatin gene (SERPINA4) is strongly correlated with a decreased risk of developing acute kidney injury in patients with septic shock [41]. Herein, we show that administration of kallistatin after onset of sepsis improved kidney and liver dysfunction in mouse models with polymicrobial infection and LPS injection. Moreover, delayed kallistatin treatment suppressed oxidative stress in the lung as indicated by reduced MDA levels in CLP mice. Oxidative stress activates inflammatory pathways and Figure 8 Proposed mechanisms mediated by kallistatin in sepsis-induced inflammation, organ injury and mortality. Kallistatin, via its heparin-binding domain, antagonizes TNF-α-and HMGB1-mediated inflammatory gene expression, and its active site is essential for inducing SOCS3 expression. HMGB1, high mobility group box-1; SOCS3, suppressor of cytokine signaling-3; TNF-α, tumor necrosis factor-α. triggers a series of events that lead to organ injury [42]. Thus, these findings indicate that kallistatin protects against sepsis-induced organ injury through reduction of oxidative stress and inflammation. In addition to preventing established sepsis-induced organ injury, kallistatin gene delivery or kallistatin protein treatment has been shown to improve survival in various animal models [23][24][25]35]. Transgenic mice overexpressing kallistatin were more resistant to Gram-negative E. coli LPS-induced mortality [23]. Moreover, kallistatin gene transfer increased survival and reduced liver and skin injury, as well as inflammation, in mice with Gram-positive streptococcal infection [24]. Furthermore, kallistatin (kallikrein-binding protein) pre-treatment at two hours before LPS injection was observed to improve the survival of endotoxemic mice [35]. Likewise, we recently reported that kallistatin pre-treatment reduced mortality in septic mice after CLP surgery [25]. Importantly, the present study demonstrates that delayed administration after onset of sepsis significantly improves the survival of mice with polymicrobial infection and endotoxemia. Therefore, kallistatin could be a promising new agent for the treatment of sepsis.

Conclusions
This study demonstrated that delayed human kallistatin treatment after onset of sepsis reduces inflammation, organ injury and mortality in both septic mouse models with polymicrobial infection and endotoxic shock. Moreover, this is the first study to indicate that kallistatin's active site plays a key role in stimulating SOCS3 expression via tyrosine kinase-PKC-ERK signaling in immune cells. As kallistatin is an endogenous molecule, kallistatin therapy may lead to the protection and/or reversal of sepsisinduced injury. The unique anti-inflammatory actions of kallistatin need to be further explored as a novel therapeutic approach for sepsis.

Key messages
Delayed administration of human kallistatin reduced organ damage, inflammation and lethality in mouse models of polymicrobial infection and lipopolysaccharide-induced endotoxemia. Kallistatin treatment attenuated established sepsis-induced inflammation and multi-organ injury in association with reduced TNF-α and HMGB1 expression, as well as increased SOCS3 synthesis. Kallistatin's active site is essential for inducing SOCS3 expression by activation of tyrosine kinase-PKC-ERK signaling. Kallistatin is a unique anti-inflammatory agent with pleiotropic actions, leading to significant protection against established sepsis.


extfieldsofstudy: Biology Medicine

text:
Vascular, but not luminal, activation of FFAR1 (GPR40) stimulates GLP-1 secretion from isolated perfused rat small intestine

Glucagon-like peptide 1 (GLP-1) plays a central role in modern treatment of type 2 diabetes (T2DM) in the form of GLP-1 enhancers and GLP-1 mimetics. An alternative treatment strategy is to stimulate endogenous GLP-1 secretion from enteroendocrine L cells using a targeted approach. The G-protein-coupled receptor, FFAR1 (previously GPR40), expressed on L cells and activated by long-chain fatty acids (LCFAs) is a potential target. A link between FFAR1 activation and GLP-1 secretion has been demonstrated in cellular models and small-molecule FFAR1 agonists have been developed. In this study, we examined the effect of FFAR1 activation on GLP-1 secretion using isolated, perfused small intestines from rats, a physiologically relevant model allowing distinction between direct and indirect effects of FFAR1 activation. The endogenous FFAR1 ligand, linoleic acid (LA), and four synthetic FFAR1 agonists (TAK-875, AMG 837, AM-1638, and AM-5262) were administered through intraluminal and intra-arterial routes, respectively, and dynamic changes in GLP-1 secretion were evaluated. Vascular administration of 10 μmol/L TAK-875, 10 μmol/L AMG 837, 1 μmol/L and 0.1 μmol/L AM-1638, 1 μmol/L AM-6252, and 1 mmol/L LA, all significantly increased GLP-1 secretion compared to basal levels (P < 0.05), whereas luminal administration of LA and FFAR1 agonists was ineffective. Thus, both natural and small-molecule agonists of the FFAR1 receptor appear to require absorption prior to stimulating GLP-1 secretion, indicating that therapies based on activation of nutrient sensing may be more complex than hitherto expected.

Introduction
Glucagon-like peptide 1 (GLP-1) is a gut hormone that has gained a central position in modern treatment of type 2 diabetes mellitus (T2DM). Currently, the therapeutic effects of GLP-1 are achieved through injectable GLP-1 analogs (GLP-1 or incretin mimetics) and oral GLP-1 enhancers, that is, DPP-4 inhibitors inhibiting enzymatic degradation of endogenous GLP-1 (Drucker and Nauck 2006). As GLP-1 is secreted from enteroendocrine L cells embedded in intestinal mucosal epithelium (Eissele et al. 1992;Orskov et al. 1996), an alternative treatment strategy is to stimulate endogenous GLP-1 release through oral Lcell secretagogues. Increased postprandial levels of endogenous GLP-1 following the bariatric procedure, Roux-en-Y gastric bypass (RYGB) (Korner et al. 2007), have already been linked to the substantial weight loss and the remission of type 2 diabetes observed postsurgery (le Roux et al. 2006;Jorgensen et al. 2012). This indicates that there is an extra capacity in the GLP-1 axis which could be mobilized through pharmacological L-cell stimulation. Physiologically, GLP-1 is secreted in response to ingestion of nutrients with dietary fat constituting a potent L-cell stimulus (Elliott et al. 1993;Beglinger et al. 2010). This secretory response may be, at least partly, mediated through activation of free fatty acid receptor 1 (FFAR1, previously GPR40)a G-protein-coupled receptor expressed on L cells (Edfalk et al. 2008;Xiong et al. 2013) recognizing long-chain free fatty acids (LCFA) released from hydrolyzed dietary fat (Briscoe et al. 2003;Kotarsky et al. 2003;Itoh and Hinuma 2005). FFAR1 constitutes an attractive antidiabetic drug target as this receptor is also expressed on pancreatic b-cells (Briscoe et al. 2003;Tomita et al. 2006;Del Guerra et al. 2010;Yashiro et al. 2012) in which FFAR1 activation potentiates glucose-stimulated insulin secretion (GSIS) (Itoh et al. 2003;Latour et al. 2007;Schnell et al. 2007;Kebede et al. 2008;Alquier et al. 2009). A number of FFAR1 selective agonists have been developed (Negoro et al. 2010;Lin et al. 2011;Houze et al. 2012;Wang et al. 2013) and several studies have confirmed the antidiabetic potential of FFAR1 activation through the direct insulinotropic effect on the b-cells (Christiansen et al. 2008;Tan et al. 2008;Lin et al. 2011;Tsujihata et al. 2011;Araki et al. 2012;Burant et al. 2012;Yashiro et al. 2012;Kaku et al. 2013).
A stimulatory effect of FFAR1 activation on GLP-1 secretion has also been reported in single cell studies and in some rodent studies (Reimann et al. 2008;Parker et al. 2009;Luo et al. 2012;Habib et al. 2013;Xiong et al. 2013), while clinical studies show inconclusive results regarding the effect of FFAR1 agonists on GLP-1 secretion (Leifke et al. 2012;Kaku et al. 2013). A detailed investigation of the direct link between FFAR1 activation and GLP-1 secretion in a physiologically relevant model has not been carried out. We, therefore, explored the effect of FFAR1 activation on GLP-1 secretion in rats using the isolated, in situ perfused small intestine. Unlike cell cultures (primary as well as cell lines) in which cells have lost their polarity, natural intercell association, and neurovascular supply, the endocrine cells in the perfused model are studied in their natural environment, allowing physiologically relevant studies of secretory mechanisms. In the present investigation, we studied dynamic changes in GLP-1 secretion in response to administration of the endogenous FFAR1 ligand, linoleic acid (LA), as well as synthetic selective FFAR1 agonists. Stimulants were administered from either the luminal or vascular side of the perfused intestine.

Ethical approval
This study was carried out with permission from the Danish Animal Experiments Inspectorate (license no.: 2013-15-2934-00833) and the local ethical committee (EMED, in accordance with the guidelines of Danish legislation governing animal experimentation.

Animals and perfusion protocol
Male Wistar rats aged 8-10 weeks were purchased from Taconic (Ejby, Denmark) and housed in pairs under a 12 h light-dark cycle with ad libitum access to water and standard chow. Nonfasted rats weighing 250 g (CV 7%) were anesthetized by subcutaneous injection with Hypnorm â /Midazolam (0.079 mg fentanyl citrate + 2.5 mg fluanisone + 1.25 mg midazolam) and placed on a heating table at 37°C. The abdominal cavity was opened with two oblique incisions and colon plus 2/3 of the small intestine were resected leaving 32 cm (CV 19%) of the proximal small intestine in situ. The vascular supply to the duodenum and pancreas was ligated and a plastic tube was inserted proximally into the gut lumen allowing a gentle flush with 37°C perfusion buffer (PB) to remove intestinal contents. A constant luminal flow of 37°C PB (0.150 mL/min) was maintained. Using a universal singlepass perfusion system (UNIPER UP-100, Hugo Sachs Elektronik-Harvard apparatus, March-Hugstetten, Germany), the gut was vascularly perfused (7.5 mL/min) via a cannula inserted into in the superior mesenteric artery. Venous effluent was collected via the cannulated portal vein. The rat was sacrificed by exsanguination immediately after vascular perfusion was established. The perfused intestine was allowed to equilibrate for 30 min before experimental protocols were initiated. Venous effluents were collected each minute and samples were immediately put on ice and stored at À20°C until analysis. As a live assessment of gut health, perfusion pressure was recorded continuously by a pressure transducer (APT300, Hugo Sachs Elektronik) and random samples of PB from both the arterial and venous side pre-and postprotocol were analyzed for respiratory parameters (PO 2 , PCO 2 , pH, and lactate) using a blood gas analyzer (ABL700, Radiometer, Copenhagen, Denmark).
In randomly selected perfusion experiments (n = 17), an approximately 1-cm midsegment of the perfused intestine was excised after termination of the experiment and stored in formaldehyde (4°C) for later histological examination (paraffin embedded and hematoxylin/eosin stained as described previously) (Kissow et al. 2012).

Stimulations
Luminal stimuli were administered as a 5 mL bolus (2.5 mL/min) followed by continuous infusion (0.150 mL/ min) throughout the remaining stimulation period. To terminate luminal stimulation, a 5 mL flush of PB (2.5 mL/ min) was administered to clear the gut lumen of stimulatory solutions. Vascular stimuli were administered intra-arterially. All experiments were completed by vascular administration of 10 nmol/L bombesin (BBS). There were no negative responders to BBS. Each perfusion experiment was executed in a fresh preparation applying either vascular or luminal stimulatory routes, respectively.

Reagents
Reagents were purchased from Sigma-Aldrich (Broendby, Denmark) unless otherwise stated. PB consisted of a modified Krebs-Ringer bicarbonate buffer containing 0.1% human serum albumin (HSA) from Millipore (Billerica, MA), 5% dextran T-70 from Pharmacosmos (Holbaek, Denmark), 3.5 mmol/L glucose, and 5 mmol/L pyruvate, fumarate, and glutamate, respectively. Prior to every experiment, PB used in the vascular perfusion circuit was added to a mixture of amino acids, Vamin â (0.5 mL/100 mL) and 3-isobutyl-1-methylxanthine (IBMX) to a final concentration of 10 lmol/L. IBMX (a phosphodiesterase inhibitor) was applied to sensitize the secretory mechanisms in the perfused gut. PB was oxygenated with a gas mixture of 95% O 2 and 5% CO 2 . FFAR1 agonists were a gift from Merck (Kenilworth, NJ) and included partial agonists, TAK-875 (Negoro et al. 2010) and AMG 837 (Hauge et al. 2014) and full agonists, AM-1638 (Brown et al. 2011Lin et al. 2012) and AM-5262 (Wang et al. 2013). FFAR1 agonists and IBMX were dissolved in dimethyl sulfoxide (DMSO) and further diluted in PB before experimental use. The perfused intestine was not exposed to DMSO concentrations above 1% (which did not significantly stimulate hormone secretion in control experiments, Fig. 3E and F, n = 6). BBS, which is a well-known GLP-1 secretagogue and thus served as a positive control, was dissolved in distilled water containing 1% HSA. The long-chain free fatty acid, LA, was administered in different formulations for vascular and luminal stimulations, respectively. For vascular stimulation, two LA formulations were tested: LA solubilized in 100% DMSO, which prior to experimental use was diluted in PB into a final LA concentration of 1 mmol/L (slightly opalescent solution), and "LA water soluble" (cat. no. L5900), that is, LA solubilized with methyl-b-cyclodextrin (MBCD), which was further diluted in PB into a final LA concentration of 100 lmol/L (clear formulation). LA for luminal stimulation was administered as sodium linoleate, "LA water soluble" or as mixed lipid micelles, respectively. Sodium linoleate powder was dissolved in isotonic saline (0.9%) into a final LA concentration of 10 mmol/L (clear formulation) by gently stirring at room temperature for 1 h and finally securing a final pH % 9.5 as described previously for sodium oleate (Beglinger et al. 2010). "LA water soluble" for luminal stimulation was dissolved in isotonic saline into a final LA concentration of 1 mmol/L (clear formulation). The lipid micelle formula was based on a previous study (Chateau et al. 2005). For preparation of 1 mL lipid micelles, 0.6 lL of 100 mmol/L oleic acid, 0.2 lL of each 100 mmol/L L-a-lysophosphatidylcholine, 100 mmol/L 2-mono-oleoylglycerol, and 25 mmol/L cholesterol were mixed in a sterile glass tube, dried under stream of nitrogen, and dissolved in 83 lL saline containing 0.1% fatty acid-free bovine serum albumin (BSA) and 24 mmol/L sodium taurocholate. Saline containing 0.1% fatty acid-free BSA was added up to 1 mL.

Statistics
Statistical evaluation was performed using GraphPad Prism 5 software (La Jolla, CA). Data are presented as mean values AE1 SEM. Statistical significance was assessed by paired t-test, comparing average hormone concentration of the initial 10-min baseline period with the initial 10 min of stimulation periods. Respective sample sizes are presented in the Results section. For intergroup comparisons of vascular stimulations with LA, TAK-875, and AMG 837, respectively, to vehicle (DMSO 1%) unpaired t-tests were performed. P-values <0.05 were considered statistically significant.

Discussion
The main finding of this study is that vascular FFAR1 activation stimulates GLP-1 secretion, whereas luminal FFAR1 stimulation is without effect in the isolated perfused rat small intestine.
In view of the therapeutic success of current GLP-1 based strategies in treatment of T2DM, the prospect to harness endogenous GLP-1 production from enteroendocrine L cells using a targeted approach has emerged . To do so, the secretory mechanisms underlying meal-induced GLP-1 release must be identified. Fat is a known stimulator of GLP-1 secretion (Elliott et al. 1993;Beglinger et al. 2010;Lindgren et al. 2011). Orlistat, an inhibitor of gastric and pancreatic lipase, has been demonstrated to attenuate postprandial GLP-1 release in humans, suggesting that hydrolysis of triglycerides into FFAs is an essential step in fat-induced GLP-1 secretion (Ellrichmann et al. 2008). Accordingly, luminal administered LCFA have been shown to stimulate GLP-1 secretion in humans (Feltrin et al. 2004;Carr et al. 2008;Beglinger et al. 2010) and rodents (Adachi et al. 2006;Dailey et al. 2010). This effect has been proposed to be mediated through activation of G-protein-coupled receptors as FFAR1 (Edfalk et al. 2008;Reimann et al. 2008;Parker et al. 2009;Xiong et al. 2013) and GPR120 (Hirasawa et al. 2005). Selective FFAR1 activation seems to be a particular attractive target for the treatment of T2DM as the receptor not only colocalizes with the incretin hormones, GLP-1, and glucose-dependent insulinotropic polypeptide (GIP) (Edfalk et al. 2008;Liou et al. 2011), but appears to be present on the pancreatic b-cells as well. Thus, selective FFAR1 activation should theoretically give rise to a robust glucose-stimulated insulin secretion (GSIS) as the b-cells would be both directly and indirectly (incretins) activated. The direct link between FFAR1 activation and GLP-1 secretion has been investigated using the GLP-1 secreting cell line GLUTag and primary L-cell cultures Habib et al. 2013). It is, however, an often neglected problem that these cells lose their natural polarity during the process of cultivation. In many tissues, polarity and contact to neighboring cells are essential for normal function. Consequently, the full functional responsiveness and secretory capacity of GLP-1-producing cells may not be preserved in these cellular models. FFAR1 activation has also been linked to GLP-1 secretion in vivo in wild-type and/or FFAR1 deficient animals (Edfalk et al. 2008;Luo et al. 2012;Xiong et al. 2013). Although in intact animals it can be difficult to distinguish direct from indirect effects, and studies of hormone secretion are hampered by the limited volume of plasma that can be harvested, particularly from mice, often in combination with lack of suitable assays. In the case of FFAR1, a tissue-specific knock out model has not yet been introduced, and since the receptor is expressed in various tissues, including the central nervous system (Briscoe et al. 2003;Nakamoto et al. 2012), pancreatic a-cells (Flodgren et al. 2007), taste buds (Cartoni et al. 2010), and other enteroendocrine cells (GIP and cholecystokinin [CCK]) (Edfalk et al. 2008), the isolated effect of receptor activation or deletion in the intestine is difficult to evaluate in vivo. The isolated, perfused small intestine allows us to study intestinal dynamics in a physiological setting where the enteroendocrine cells maintain natural polarity and integration in an intact intestinal mucosa. In addition, the intestine is isolated from the systemic circulation which provides the means to investigate organ-specific responses without influences from whole body regulatory mechanisms. Isolated perfused small intestines have, to the best of our knowledge, not yet been used to approach the involvement of FFAR1 activation in GLP-1 secretion.
Using this particular method, we hereby show that vascular, but not luminal, FFAR1 activation significantly stimulates GLP-1 secretion. This was evident using the endogenous ligand, LA, as well as the synthetic agonists.
Intra-arterial infusion of LA significantly stimulated GLP-1 release compared to basal levels ( Fig. 1C and D), while sodium linoleate administered luminally only elicited a transient nonsignificant peak in GLP-1 secretion ( Fig. 1A and B). An intergroup comparison between vascular LA and the DMSO control suggested that the observed effect of vascular LA may have been attributed to DMSO. However, an evident GLP-1 response was also observed upon vascular, but not luminal, stimulation with LA + MBCD (Fig. 2), which strongly supports a vascular LA-induced FFAR1 activation. The absence of response to intraluminal LCFA is consistent with a previous study on isolated perfused rat small intestine, where 100 mmol/L of luminal oleate did not increase GLP-1 secretion (Dumoulin et al. 1998).
The finding that vascular and not luminal LA stimulates GLP-1 secretion contrasts with previous results from an in vivo rodent study, demonstrating increased GLP-1 plasma levels after intrajejunal infusion of LA (Dailey et al. 2010). However, in this particular study, GLP-1 plasma levels were measured as active hormone in trunk blood from sacrificed animals after a 5-day continuous intestinal LA infusion. As the active isoforms of GLP-1 have an ultrashort half-life (less than 2 min in humans and less in rodents) and nutrient-induced GLP-1 secretion should be measured postprandially rather than after chronic stimulation, these findings cannot be considered physiologically relevant. Clinical studies have shown increased GLP-1 secretion in response to oleate in some (Beglinger et al. 2010), but not in all studies (Hansen et al. 2011). In addition, GLP-1 secretion after oleate infusion may result from activation of neuroendocrine loops involving vagal nerve activity and secretion of CCK and GIP, which both have been shown to stimulate GLP-1 secretion (Fieseler et al. 1995;Rocca and Brubaker 1999).
In the present study, increased GLP-1 secretion was also observed upon vascular FFAR1 activation using four synthetic selective FFAR1 agonists (Figs. 3 and 4). The partial FFAR1 agonists, TAK-875 (Negoro et al. 2010) and AMG 837 (Lin et al. 2011), both elicited a similar, modest, yet significant, GLP-1 response upon vascular administration compared to basal levels ( Fig. 3A-D), while luminal administration did not affect GLP-1 secretion (Fig. 5). Intergroup comparison to the DMSO control showed that only the GLP-1 response induced by vascular AMG 837 was significantly different from vascular vehicle. This is consistent with very recent findings demonstrating a modest effect of AMG 837 and TAK-875 on GLP-1 secretion in vitro (Hauge et al. 2014), while only little or no effect was seen after oral gavage in mice in vivo Hauge et al. 2014). Clinical studies investigating the antidiabetic potential of TAK-875 showed inconclusive effects on postprandial GLP-1 plasma levels (Leifke et al. 2012;Kaku et al. 2013). In the present study, the full FFAR1 agonists, AM-1638 ) and AM-5262 (Wang et al. 2013), both caused a pronounced GLP-1 response, at least comparable to the response induced by intraluminal glucose (Fig. 4A-F). The efficacy of AM-1638 and AM-5262 Wang et al. 2013) may be a consequence of a combined G s and G q signaling activated by these agonists rather than G q signaling alone (Hauge et al. 2014).
The dynamic measurements in the present study, surprisingly, revealed that GLP-1 secretion did not return to baseline after termination of infusion with 1 lmol/L AM-1638 and AM-5262, respectively (Fig. 4C-F). At 0.1 lmol/L AM-1638, a defined GLP-1 response within the infusion period was observed ( Fig. 4A and B) which may merely suggest that high agonist concentrations require long wash out periods. Luminal administration of AM-1638 did not affect GLP-1 secretion ( Fig. 5E and F), but as mentioned above luminal stimulation with AM-5262 at a 10-fold higher concentration (10 lmol/L) than that causing a dramatic vascular stimulation produced a small, but significant, prolonged response (Fig. 5G, H). We interpreted this as a response to absorption of the agonist from the luminal and into the interstitial/vascular compartment.
In order to verify that the absence of response to luminal FFAR1 stimulation was not due to mucosal damage in luminally stimulated gut segments, mucosal integrity was confirmed by subsequent histological examinations of the perfused intestinal segments (data not shown). Furthermore, as a positive luminal control, we administered a 20% (w/v) luminal glucose solution, which is considered a robust stimulus of GLP-1 secretion and has previously been shown to induce a strong GLP-1 response from perfused rat small intestines (Dumoulin et al. 1998;Kuhre et al. 2014b). Consistent with these studies, intraluminal glucose infusion caused a pronounced GLP-1 response ( Fig. 1E and F). Moreover, luminal administration of protein hydrolysates has recently been shown to stimulate GLP-1 secretion in the same preparation (Svendsen et al. 2014). However, LCFA may interact differently from glucose and protein hydrolysates with the luminal intestinal milieu due to their lipophilic nature. To exclude lack of solubility of LCFA as an explanation for the lack of GLP-1 secretion upon luminal FFAR1 activation, different solubilization methods were tested in pilot experiments. In addition to the administration of luminal lineolate (Fig. 1A), we explored luminal stimulation with LA solubilized with MBCD. Despite increasing GLP-1 secretion upon vascular administration ( Fig. 2A), this formulation did not stimulate GLP-1 secretion when administered intraluminally (Fig. 2B). Finally, to mimic the complex physiological intestinal transport of LCFA, luminal stimulation with mixed lipid micelles was investigated. This luminal stimulus also did not increase GLP-1 secretion (Fig. 2C).
Based on these futile efforts to activate GLP-1 secretion upon intraluminal administration of LA solutions, we conclude that it is not a hindered interaction of lipophilic luminal stimulants with FFAR1 that underlies the lack of GLP-1 response to luminal FFAR1 activation. Rather our findings could point to a predominant expression of FFAR1 in the basolateral L-cell membrane facing the intercellular compartment and the blood stream. In this case, physiological LCFA-induced GLP-1 secretion may occur secondary to intestinal lipid absorption. Supporting this notion, intraluminal lipid stimulation along with pharmacological inhibition of enterocyte formation of chylomicrons (Lu et al. 2012) and lipoproteins (Hata et al. 2011) resulted in reduced lipid-induced GLP-1 2015 | Vol. 3 | Iss. 9 | e12551 Page 10 secretion. Identification of the exact membrane localization of L-cell FFAR1 would require advanced histological examination which was beyond the scope of this study.
Thus, it appears that L-cell receptor activation not only requires absorption of the ligands, but possibly also appearance in the blood stream.
In conclusion, in order to develop pharmacological strategies for L-cell stimulation, we need to clarify the secretory mechanisms underlying nutrient-induced GLP-1 secretion. The isolated perfused small intestine is a robust model to explore such mechanisms. Because of the preserved polarity of the perfused preparation, we were able to demonstrate that vascular rather than luminal activation of FFAR1 stimulated GLP-1 secretion. Although our findings are consistent with the notion that oral FFAR1 agonists may constitute future L-cell stimulants for T2DM and obesity treatment, they also show that the mechanisms involved are more complicated than anticipated and appears to require appearance of the FFAR1 ligands in the circulation.


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Methylene Blue Protects Astrocytes against Glucose Oxygen Deprivation by Improving Cellular Respiration

Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration.

Introduction
The brain is almost exclusively dependent on an uninterrupted supply of glucose and oxygen in order to generate energy to meet its physiological requirements. Due to this high energy demand and lack of energy reserves in the brain, short disturbances in the availability of oxygen and glucose can quickly impair energy production and cause metabolic failure [1]. Impaired cerebral metabolism is one of the major features of various neurological disorders including Alzheimer's disease [2], neuroglycopenia [3] and especially ischemic stroke [1,4]. During ischemic stroke, interruption of cerebral blood flow dramatically reduces the availability of oxygen and glucose to brain cells. Consequently, within minutes of glucose and oxygen depletion cellular ATP production is severely hampered. This compromises bioenergetic metabolism [5] and energy-dependent processes, such as ionic homeostasis and reuptake of excitatory amino acids, resulting in excitotoxicity, cytotoxic edema and, ultimately, cell death [6]. With thrombolytic therapy, reperfusion of the ischemic brain can be achieved in eligible stroke patients. However, in some circumstances, reperfusion does not reestablish physiological energy metabolism but further deteriorates the injured brain [7,8] by increasing reactive oxygen species (ROS) production [9] and secondary waves of excitotoxicity [1,10]. Therefore, a vascular approach to restoring energy substrates as a therapeutic intervention is greatly limited for the treatment of ischemic stroke. A cellular approach to maintenance of bioenergetic metabolism might provide additional treatment for ischemic stroke patients.
Methylene Blue (MB) was synthesized in 1876 and has been used to treat methemoglobinemia, cyanide poisoning and malaria [11]. MB has been shown to attenuate mitochondrial dysfunction under stress [12], increase mitochondrial complex IV activity [13,14], and delay senescence of human fibroblasts in vitro [13]. The unique redox nature of MB enables it to shuttle electrons directly from mitochondrial complex I to Cytochrome C, bypassing complex I/III blockage, thereby preventing electron leakage and superoxide production, and improving mitochondrial respiration [14][15][16]. Indeed, MB has been shown to improve cerebral blood flow, increase glucose uptake and the cerebral metabolic rate of oxygen consumption under normoxia and hypoxia in vivo [16]. MB has been recently studied in various CNS disorders and reported to be protective in rodent models of ischemic stroke [17], Parkinson's disease [22], Alzheimer's disease [18,19], and traumatic brain injury [20].
Astrocytes are the most abundant cells in mammalian brain vastly outnumbering neurons [21]. They possess a unique cyto-anatomical and phenotypic attributes that optimally positions them around neurons and vessels. Astrocytes also play a key role in neurovascular interaction coupling neuronal activity and cerebral blood flow (CBF), which facilitates an uninterrupted supply of oxygen and glucose to meet the high metabolic demands of neurons [22,23]. In addition, there is increasing evidence indicating that astrocytes are active players in brain energy production, utilization, and storage via astrocyte-neuron metabolic coupling [24,25]. Astrocytes have been shown to support neuronal metabolism by producing lactate through glycolysis and activation of glycogen catabolism [26]. This neurovascular and metabolic coupling appears to play an important role in the control of neuronal activity in the brain. Impairments in astrocyte metabolism are increasingly being recognized as important contributors to neuronal dysfunction and neurodegenerative processes [27][28][29]. The goal of the current study was to test the hypothesis that MB protects astrocytes against oxygen glucose deprivation & reoxygenation induced cell death by improving cellular respiration.

Primary astrocyte cultures
All procedures performed during preparation of primary astrocyte cultures from C576bL6 mice were approved by the Institutional Animal Care and Use Committee of the University of North Texas Health Science Center (UNTHSC) Fort Worth. Primary astrocyte enriched cultures were prepared from 1 day old C57BL6 pups as previously described [30]. Briefly, day old mouse pups were anesthetized by hypothermia followed by decapitation with sharp surgical scissor. Under aseptic conditions the cerebral cortices were dissected and meninges were removed. Cortical tissue was then digested in 0.25% trypsin (Sigma) at 37°C for 20 min. A single cell suspension was prepared by repeated pippeting of the digested cortical tissue through 3 different bore sized Pasteur Pipette. The cell suspension was strained through 40 μM size cell strainer and plated in a 10 cm culture plate in high glucose Dulbecco's Modified Eagle's Medium, (DMEM with 4500 mg/l Glucose, 4 mM l-glutamine, 1 mM sodium pyruvate, Thermo Scientific) containing 10% Fetal Bovine serum and Streptomycin (10,000 μg/ml)-Penicillin (10,000 units/ml) and cultured in a humidified incubator at 37°C a with 5% CO 2 for two weeks. Once the cultures plates became 90% confluent, the plates were constantly shaken for 48hr in a CO 2 incubator at 37°C to eliminate microglia and other types of cell contaminants.
In Vitro oxygen glucose deprivation model Astrocytes were cultured in DMEM (11 mM Glucose, 1 mM pyruvate, 4 mM L-glutamine, Thermo Scientific) for 3 days before experiment. For OGD, the cells were washed twice with sterile Phosphate Buffer Saline (PBS) and incubated in glucose, pyruvate and FBS free DMEM (Thermo Scientific) for 6hr in an anaerobic chamber with 0.1% O 2 and 5% CO 2 at 37°C. During reoxygenation, astrocyte media was supplemented with D-Glucose (11 mM), pyruvate (1 mM) and with different concentrations of MB and incubated in 5% CO 2 , 95% air at 37°C for 24hr. At the end of the experiment the viability of astrocytes was determined by Calcein AM assay using a Tecan infinite M200 plate reader and images were taken with Zeiss Observer Z1 microscope (Fig 1).

Oxygen consumption rate
Oxygen consumption rate was determined as previously described [31]. Briefly, primary astrocytes were seeded in a XF24 plate at a density of 60,000 cells/well and cultured for 2 days to form an even monolayer. On the day of the experiment the culture media was replaced with XF24 media and incubated for 1hr in a non CO 2 incubator at 37°C. In the accompanying cartridge of XF24 plate provided in the assay 100X concentration of Rotenone (final concentration 100 nM), FCCP (final concentration 300 nM), Oligomycin (final concentration 1 μg/ml), and MB (final concentration 0.1, 1 and 10 μM), were loaded. The XF24 plate and cartridge were loaded into Seahorse Bioscience XF24 Extracellular Flux Analyzer and drugs were injected at specified time points and oxygen consumption rate was determined (Fig 2). Protein concentration of each sample was determined using Pierce 660 nm Protein assay reagent (Thermo Scientific) and was used to normalize respective oxygen concentration rates.

Fluorescence-lifetime imaging microscopy (FLTIM)
Astrocytes were cultured on a 35 mm cell culture dish with a cover glass in DMEM (11 mM Glucose, 1 mM pyruvate, 4 mM L-glutamine, Thermo Scientific) with 10% FBS for 2 days. For FLTIM the astrocytes were incubated in Tris (2, 2 0 -bipyridyl) dichlororuthenium (II) hexahydrate (120 μM in PBS, Sigma) for 2hr. The cells were then washed twice and incubated in sterile Dulbecco's Phosphate Buffered Saline (DPBS) with 10 mM Dextrose (Sigma). MB and of glucose oxidase (GO, 5 units/mL) were diluted in DPBS from respective stock solutions and added to the dishes during microscopy. Time resolved images were obtained on a confocal Micro-Time 200 (Picoquant GmbH, Berlin) system (Fig 3). Excitation was provided from a 470 nm pulsed diode laser (PicoQuant) operating at a 320 kHz repetition rate, and it was reflected off of a 490 nm dichroic plate into an Olympus IX71 inverted microscope. The light passed through an Olympus 60X 1.2 NA objective, and the collected fluorescence was filtered by a 488 nm long wave pass, interference filter before passing through a 50 μm confocal pinhole. The light path terminated at a hybrid photomultiplier assembly detector, also from PicoQuant. The time response of this detector, including the width of the laser pulse, was estimated to be less than 120 ps. The signal from the detector was routed into a PicoHarp 300 (PicoQuant) time correlated single photon counting (TCSPC) module which time-tagged each photon with a resolution of 512 ps per bin. Fluorescence decay curves were analyzed using a single component, exponential, tail-fitting routine which was performed by the software SymPhoTime, v. 5.3.2 from PicoQuant (Fig 3).

Superoxide analysis
Mitochondrial superoxide production in the astrocytes was determined according to manufacturer's instruction using MitoSOX (Molecular Probes). Astrocytes cultured for 24hrs were washed once with sterile PBS and treated with either MB or Antimycin-A (50 μM) for 20 min. At the end of 20 min astrocytes were washed once with PBS and incubated with MitoSOX (5μM) for 10 min at 37°C. MitoSOX fluorescence was quantified using a Tecan infinite M200 plate reader (Excitation/emission maxima:~510/580 nm) and images were taken with Zeiss Observer Z1 microscope (Fig 4).

Glucose uptake
Glucose uptake in astrocytes was determined using glucose analog 2-NBDG as previously described [16]. Briefly, astrocytes were cultured in 96 well culture plates (5000 cells/well) and 25 mm cover slip (30,  buffer and glucose uptake in astrocytes in 96 well plates was determined using a Tecan Infinite M200 plate reader (Excitation/Emission for 2-NBDG~465/540 nm). The coverslips were used for microscopy using Zeiss Observer Z1 microscope (Fig 5).

Hexokinase activity
Activity of hexokinase (HK) was measured at 37°C in a Flexstation (Molecular devices) as described earlier with minor modification [32]. The assay principle involved enzymatic conversion of glucose to glucose 6-phosphate by hexokinase which is further converted to gluconate 6-phosphate by glucose-6-phosphate dehydrogenase during which one micromole of NAD + is reduced per one unit activity/min. The change in absorbance is then measured using a spectrophotometer. For the assay, 0.67 M Glucose, 16.5 mM Adenosine 5'Triphosphate, 6.8 mM NAD and 300 IU/ml of glucose-6-phosphate dehydrogenase were prepared in 0.05 M Tris Ã HCl buffer, pH 8.0 with 13.3 mM MgCl2. In a 96 well plate, to each well 228 μl of Tris MgCl2 buffer, 50 μl of glucose and 10 μl each of ATP, NAD and glucose-6-phosphate dehydrogenase were added. Finally 10 μl of cell lysate was added and the change in absorbance was determined at 340 nm. ΔA/min was then calculated from initial linear portion of the curve (Fig 5C). Protein concentration of the samples was determined by colorimetric analysis using Pierce 660 nm Protein assay reagent (Thermo Scientific) and Tecan Infinite M200 plate reader. Enzyme activities were expressed as units per milligram of protein (Fig 5).

ATP assay
The change in cellular ATP levels was determined using ATP kit (Invitrogen) as previously described [31]. Briefly, the astrocytes were cultured for 24hr in a 6 well culture plate at a density 2.5 X 10 4 cells/well in DMEM (11 mM Glucose, 1 mM pyruvate, 4 mM L-glutamine, Thermo Scientific) at 37°C. The cells were washed twice with glucose and pyruvate free KRH buffer and incubated in KRH buffer for 30 min. The KRH buffer was replaced with culture medium (DMEM) with different concentrations of MB and incubated for either 30 or 60 min. The cells were then detached with trypsin (Sigma) and washed twice with PBS and lysed with 100 μl of ATP assay buffer (500 mM Tricine buffer, pH 7.8, 100 mM MgSO 4 , 2 mM EDTA, and 2 mM sodium azide, 1% Triton X-100). ATP reaction buffer (100 μl, 30 μg/ml D-luciferin, 20 μM DTT, and 25 μg/ml luciferase) was added to 10 μl of cell lysate and the luminescence was measured using a Tecan Infinite M200 plate reader. The ATP values were determined from a standard curve and normalized to the protein content of each sample (Fig 6).

Western blot analysis
At the end of OGD-reoxygenation the cells were lysed in a cell lysis buffer (20 mM of Tris, 100 mM NaCl, 1 mM DiSodium-EDTA, 0.5% Triton-X) with protease and phosphatase inhibitors (1:100). Protein content of the samples was determined using Pierce 660 nm Protein assay reagent (Thermo Scientific). The samples were resolved on 10% SDS gel and transferred to a nitrocellulose membrane. The membranes were incubated overnight in primary antibodies against glycogen synthase (1:500; rabbit, Cell Signaling), Phospho-glycogen synthase (1:500; rabbit, Cell Signaling), and actin (1:1000; mouse, Santa Cruz) followed by secondary antibody (Jackson Immunoresearch, West Grove, PA). Using Biospectrum 500 UVP imaging system chemiluminescence signal was detected (Fig 6). The protein densities of glycogen synthase, Phospho-glycogen synthase were normalized to actin density.

Glycogen Analysis
Total glycogen in astrocytes was determined by using Abcam Glycogen assay kit according to the manufacturer's instruction. For sample preparation following OGD-reoxygenation with or without MB, the astrocytes were detached with 0.25% trypsin (Sigma), washed twice with PBS and resuspended in 150 μl of double distilled water. The cells were lysed by sonication and boiled for 5min at 95°C. In a 96 well plate, 20 μl of sample was incubated in glycogen hydrolysis buffer containing glucoamylase enzyme provided with the kit for 30 min at room temperature. After incubation, OxiRed Probe, glycogen hydrolysis enzyme mix, glycogen development enzyme mix were added to samples and incubated for another 30 min. The glycogen content was determined by using fluorometric assay (excitation/emission = 535/587 nm) and the fluorescence was measured using Tecan Infinite M200 plate reader. Corrections for background glucose were made in all the samples and the corrected fluorescent readings were applied to standard curve and amount of glycogen in each well was determined (Fig 6). The glycogen content of each sample was normalized to their respective protein content and plotted using Graph Pad Prism 5.

Statistical analysis
Statistical analysis was performed using Graph Pad Prism 5. Results are expressed as mean ± standard error of mean (SEM). When comparing two groups a t-test was used to identify any significant differences. For comparison of multiple groups, two-way analysis of variance was used and post-hoc Bonferroni analysis was done to identify the significant differences. p < 0.05 was considered as statistically significant.

MB protects astrocytes against transient OGD-reoxygenation induced cell death
In vitro 6hr OGD and 24hr reoxygenation caused a significant cell death (50%) in primary astrocytes and MB treatment during OGD did not confer any protection, rather increased cell death induced by OGD (Fig 1). However, when administered immediately after reoxygenation, MB conferred a significant (p<0.05%) protection against transient OGD induced cell death (Fig 1).

MB increases oxygen consumption rate in astrocytes
We determined oxygen consumption rate (OCR) in astrocytes by sequential injection of MB/ vehicle, oligomycin, FCCP and rotenone. Our results showed that MB and vehicle treated astrocytes had significantly different OCR at all-time points of the experiment. MB (10 μM) administration induced a significant increase in OCR in astrocytes compared to vehicle treated cells (Fig 2). The results obtained using the Seahorse Bioscience XF24 Extracellular Flux analyzer indicated the changes in oxygen concentration in the media. We further determined the effect of MB treatment on intracellular oxygen using an oxygen sensing dye, Tris (2, 2 0 -bipyridyl) dichlororuthenium (II) hexahydrate and FLTIM. Ruthenium's (II) fluorescence is quenched in the presence of high oxygen concentration [33]. Therefore, the lifetime of ruthenium fluorescence provides an indirect measurement of intracellular oxygen concentration. FLTIM indicated that the average fluorescence lifetime of ruthenium was significantly reduced following MB treatment as compared to vehicle control (Fig 3), indicating that MB treatment significantly increased oxygen flux into astrocytes in agreement with the increased OCR as observed in Seahorse Flux analyzer experiment. The sensitivity of Ruthenium (II) to cellular oxygen status was confirmed by glucose oxidase (GO), which consumes intracellular oxygen, hence, creates a low intracellular oxygen tension and increases average lifetime of Ruthenium (II)'s fluorescence. During reoxygenation, perturbation of mitochondrial electron transfer chain (ETC) results in overproduction of superoxide [34]. We further investigated the effect of MB on superoxide production using Mitosox assay. We found that MB treatment did not increase superoxide production, while, as predicted, antimycin-A significantly increased astrocytes superoxide production (Fig 4).

Methylene Blue increases glucose uptake and restore hexokinase activity after OGD in astrocytes
To determine if MB increases glucose uptake in astrocytes we used 2-NBDG, a fluorescent glucose analogue, which cells take up via glucose transporters. Once taken up, 2-NBDG is metabolized very slowly. This enables quantification by measuring its fluorescence intensity. Fluorescent microscopy and quantitative analysis of 2-NBDG uptake revealed that MB increased glucose uptake in astrocytes in a concentration dependent manner (Fig 5). MB significantly increased glucose uptake in astrocytes by 89%, 200% and 225% at the concentrations of 0.1 μM, 1 μM and 10 μM, respectively.
Hexokinase is the rate limiting enzyme in glycolysis which phosphorylates glucose and traps it for cellular utilization. We observed the effect of MB on hexokinase activity in astrocytes following OGD insult. Our results showed that following transient OGD there was a significant reduction of hexokinase activity in astrocytes which was significantly abolished by MB treatment (Fig 5).

MB promotes ATP production and glycogen synthesis in primary astrocytes
We determined ATP production in astrocytes following MB treatment. MB significantly increased ATP production in astrocytes following 60 min treatment in normoxic condition (Fig 6). Glycogen is a high molecular-weight glucose polymer which is used as energy storage in the brain. We observed the effect of MB on glycogenesis in astrocytes. Glycogen synthase (GS) is the enzyme involved in the conversion of glucose to glycogen. This reaction is inhibited by phosphorylation of GS. Our Western blot analysis indicated that transient OGD followed by reoxygenation inhibited glycogenesis by decreasing GS which was attenuated by MB treatment (Fig 6). This result was further confirmed by analysis of total glycogen in astrocytes. Results from glycogen analysis showed that glycogen content in astrocytes following OGDreoxygenation was significantly less compared to normoxia control and that MB treatment significantly increased glycogen content in astrocytes after OGD (Fig 6).

Discussion
In the current study we determined the protective role of MB against energy deprivation in primary astrocytes and delineated its underlying mechanism. Using an in vitro model of transient OGD, which resembles ischemia reperfusion injury, we demonstrated that MB is protective in astrocytes against energy deprivation-induced cell death when administered immediately after restoration of glucose and oxygen.
Ischemic stroke causes a reduction of cerebral blood flow, resulting in ischemic brain damage. Prompt restoration of blood flow is crucial to reduce or prevent neurological damage in ischemic stroke patients. Paradoxically, reperfusion can exacerbate brain damage through reperfusion injury [35]. Most notably, electrons leaking from the electron transport chain (ETC), mostly at complex I and complex III, react with oxygen to yield superoxide anion, which can be converted into other ROS [36,37]. With the primary ETC complexes damaged by ischemia, restoration of oxygen after reperfusion leads to a surge in ROS production which in turn facilitates a vicious cycle of accelerated mitochondrial damage, excitotoxicity, lipid peroxidation, and inflammation [38]. Oxidative stress is the essential mechanism for ischemia/reperfusion and has been proposed as the key culprit for ischemic stroke damage [39]. However, the failure of the SAINT-II trial has raised concern regarding the free radical scavenger approach for the treatment of ischemic stroke [39,40]. Our current study indicates that MB differs from the traditional antioxidants in that its protective action was only afforded when administered after reoxygenation. When administered during glucose and oxygen depletion, MB in fact exaggerates the damage. The identified unique protective action of MB against OGD-reoxygenation damage in astrocytes is in line with our previous studies that MB functions as an alternative mitochondrial electron carrier and enhances mitochondrial oxidative phosphorylation [14,15].
Our current study demonstrates that MB significantly increases oxygen influx and oxygen consumption rate in astrocytes, indicating that MB enhances astrocyte glucose catabolism and mitochondrial oxidative phosphorylation. Glucose is the principal and obligatory fuel of the mammalian brain required for high energy metabolism. In the event of glucose oxygen deprivation as seen during ischemia, subsequent loss of ATP can quickly lead the brain tissue to irreversible metabolic failure. In the present study we demonstrated that MB significantly increased glucose uptake in astrocytes in a concentration dependent manner. Importantly, the increase in glucose uptake induced by MB is accompanied by an increase in hexokinase activity. Hexokinase is the first enzyme in glucose metabolism and is responsible for irreversible conversion of glucose to glucose-6-phosphate. The increase in hexokinase activity induced by MB indicates that MB indeed enhances glucose metabolism. In astrocytes an increase in ATP production was consistently observed upon MB treatment. Besides its crucial function in the glycolytic pathway, hexokinase also plays an important role in the cellular survival mechanism by interacting with mitochondrial BCl-2, an antagonist of cell death (BAD) and modulates apoptosis in response to glucose deprivation. Hexokinase isoforms especially HK II is known to elicit anti-apoptotic function and protect astrocytes and neurons from hypoxia. The present study demonstrates a significant reduction in hexokinase activity following OGD in astrocytes which could lead to reduction in glucose utilization. MB treatment prevents the reduction and maintains the hexokinase activity which improves glucose metabolism following OGDreoxygenation.
The greatly impaired cerebral ATP production during ischemia is not completely restored even after tissue reperfusion and reduction in oxidative metabolism of glucose continues to persists in later stage of reperfusion [41,42]. In our study we observed that MB increases astrocyte glucose uptake, hexokinase function, and oxygen consumption as well as ATP production. These effects are particularly beneficial in the post ischemic brain which primarily depends on glycolysis for ATP production and in turn causes acidosis which is detrimental to the surviving brain cells [43,44]. We speculate that the bioenergetic action of MB is a direct result of its alternate mitochondrial electron carrier action, which bypasses the mitochondrial complex I-III blockage thus improving mitochondrial respiration [15]. This notion is further supported by the detrimental action of MB against ischemia reperfusion injury when administered during OGD. During ischemia, acceleration of oxygen consumption and glucose metabolism might further exaggerate ischemic damage. However, the deleterious effect of MB on ischemia when administered during OGD should not be interpreted as a contradiction for using MB before thrombolytic therapy in ischemic stroke patients. MB might be limited in its ability to reach the ischemic territory without thrombolysis induced reperfusion. Indeed, a recent study demonstrated that MB treatment delays progression of perfusion-diffusion mismatch to infarct in a permanent ischemic stroke model [45].
In the current study, we observed that ATP production was improved in astrocytes in response to MB treatment. However, the increase in ATP upon MB treatment was not in proportion to the increase in glucose uptake and hexokinase activity. Mitochondrial complex I and III serve as the major proton pumps which generate a proton gradient across the mitochondrial inner membrane, the ultimate driving force for mitochondrial ATP production. We speculate that, as an alternative mitochondrial electron carrier bypassing complex I-III, MB might have a partial mitochondrial uncoupling effect which renders a disproportional increase of ATP production in relation to the increase of glucose uptake and hexokinase activity after MB treatment. In addition, the disproportional increase of ATP production might be due to the increase of glycogenesis upon MB treatment. Glycogen, a polymer of glucose, is exclusively stored in adult brain astrocytes [46] and it has been reported that almost 50% of brain glucose is taken up by astrocytes under physiological conditions [47]. Studies have indicated that astrocyte glycogen stores can preserve neuronal function and viability in the conditions of restricted ATP availability [24,25,48]. We found a significant decrease of glycogenesis in astrocytes following glucose and oxygen deprivation evidenced by reduction of both active unphosphorylated glycogen synthase and glycogen content in astrocytes which was attenuated by MB treatment. Therefore, the enhancement of glycogenesis and glycogen stores might also contribute the disproportional increase of ATP production and glucose uptake upon MB treatment. As mentioned above, ischemia induced injury at cellular level is reflected as severe derailment of cellular metabolic processes. This impaired process is not restored even after reperfusion as a result the cells undergo apoptosis leading to growth of infarct during chronic stages of injury. Our study indicated that administration of MB early during reoxygenation alleviated the bioenergetic deficiency and we presume this caused the cells to reestablish metabolic integrity which in long term prevented metabolic failure induced cell death.
In summary, we demonstrated that MB exerts a unique protective effect in astrocytes against transient oxygen glucose deprivation-reoxygenation induced cell death. MB provides protection when administered during reoxygenation, while it exaggerates OGD-reoxygenation damage when administered during OGD. In addition, MB enhances astrocyte glucose metabolism and mitochondrial oxidative phosphorylation, increasing astrocyte energy storage. The unique protective action of MB in astrocytes might provide a novel cellular approach to maintaining brain bioenergetic metabolism as a combined therapy with thrombolysis for the treatment of ischemic stroke.


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Swertianlarin, an Herbal Agent Derived from Swertia mussotii Franch, Attenuates Liver Injury, Inflammation, and Cholestasis in Common Bile Duct-Ligated Rats

Swertianlarin is an herbal agent abundantly distributed in Swertia mussotii Franch, a Chinese traditional herb used for treatment of jaundice. To study the therapeutic effect of swertianlarin on cholestasis, liver injury, serum proinflammatory cytokines, and bile salt concentrations were measured by comparing rats treated with swertianlarin 100 mg/kg/d or saline for 3, 7, or 14 days after bile duct ligation (BDL). Serum alanine aminotransferase (ATL) and aspartate aminotransferase (AST) levels were significantly decreased in BDL rats treated with swertianlarin for 14 days (P < 0.05). The reduced liver injury in BDL rats by swertianlarin treatment for 14 days was further confirmed by liver histopathology. Levels of serum tumor necrosis factor alpha (TNFα) were decreased by swertianlarin in BDL rats for 3 and 7 days (P < 0.05). Moreover, reductions in serum interleukins IL-1β and IL-6 levels were also observed in BDL rats treated with swertianlarin (P < 0.05). In addition, most of serum toxic bile salt concentrations (e.g., chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA)) in cholestatic rats were decreased by swertianlarin (P < 0.05). In conclusion, the data suggest that swertianlarin derived from Swertia mussotii Franch attenuates liver injury, inflammation, and cholestasis in bile duct-ligated rats.

Introduction
Accumulation of toxic bile acids in hepatocytes and the amplification of inflammation in cholestasis are the major factors resulting in liver injury and fibrosis [1][2][3]. Multiple pathologies, that is, gallstone obstruction of the bile duct, biliary atresia, pancreas tumors, and drug toxicity, can cause persisting cholestasis which can lead to liver failure, fibrosis, cirrhosis, and death [4][5][6]. Ursodeoxycholic acid (UDCA) and its analogs can inhibit inflammation and enhance elimination of toxic bile acids. It is the only approved drug that is widely clinically used to treat cholestatic patients such as primary biliary cirrhosis (PBC) [7,8]. However, one-third to two-thirds of cholestatic patients with PBC do not completely respond to UDCA [9,10]. INT747, an FXR agonist, is a Phase II studies drug that exerts anticholestatic effects by altering bile acids metabolism in experiment models [11][12][13][14]. Therefore, the search for potential drugs that target inflammation and bile acids pools for the treatment of cholestasis is important.
Swertianlarin is an iridoid compound that is present in Swertia davidi Franch and Swertia mussotii Franch [15][16][17][18]. These two types of Swertia Franch are the traditional herbs used for the treatment of jaundice caused by viral hepatitis. It has been used for many centuries in southwest China and is supposed to protect the liver from injury [15,18], 2 Evidence-Based Complementary and Alternative Medicine have anti-inflammatory properties, and ameliorate cholestasis. Swertianlarin from Enicostemma axillare is known to have antioxidant and hepatoprotective effects against Dgalactosamine-induced acute liver damage in rats [19]. Previous studies have also reported that swertianlarin inhibits inflammation in adjuvant-induced arthritis and in interleukin IL-1 -induced rat fibroblast-like synoviocytes [20][21][22][23]. However, the anti-inflammation effect of swertianlarin on cholestasis remains to be clarified. Furthermore, whether swertianlarin alters the bile acid pool and reduces liver injury on cholestasis is unclear.
To address whether swertianlarin derived from Swertia mussotii Franch attenuates inflammation and liver injury on cholestasis, we assessed the liver injury, serum proinflammatory cytokine levels, and the concentrations of serum bile acids in a bile duct-ligated rat model treated with swertianlarin.

Chemicals. Swertianlarin, isolated from Swertia mussotii
Franch as reported previously [24], was kindly provided by Chongqing Academy of Chinese Material Medical with a purity of 98% as analyzed by HPLC. All other chemicals were of analytical grade and purchased from Sigma-Aldrich (Sigma-Aldrich Chemical Co., St. Louis, MO, USA).

Animals and Treatments.
The male Sprague-Dawley (SD) rats were provided by the Center of Laboratory Animals of Third Military Medical University, Chongqing, China. All experimental protocols were approved by the Ethics Committee of Third Military Medical University. Efforts were made to reduce animal suffering and minimize animal number used. Animals were housed in plastic cages with free access to food and water under a 12 h light/dark cycle. The normal rats were given swertianlarin in 4 groups: 0, 10, 50, and 100 mg/(kg/d), dissolved in 1% Tween-20 saline, = 7, by gavage for 14 days. All rats survived and the serum biochemistries alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acids (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL) were normal in the study (data not shown). In this study, rats were randomly divided into three groups ( = 7 per group): sham saline, BDL without swertianlarin, and BDL with swertianlarin with each containing from five to seven animals. In the BDL swertianlarin group, rats were pretreated with swertianlarin 100 mg/(kg/d) dissolved in 1% Tween-20 by gavage for 1 d and then underwent BDL followed by continued administration of swertianlarin for 3, 7, and 14 d. In the BDL saline groups, rats underwent BDL after pretreatment with only 1% Tween-20 saline by gavage for 1 d and then continued treatment with only 1% Tween-20 saline for 3, 7, and 14 d. In the sham saline control group, rats were pretreated with 1% Tween-20 saline by gavage for 1 d and underwent a sham-operation followed by only 1% Tween-20 saline for 3, 7, and 14 d. After 3, 7, and 14 d, animals were sacrificed randomly between 9:00 am and 11:00 am. Blood was placed on ice for 60 min and centrifuged (8000 g, 10 min) to prepare serum. The serum was immediately stored at −80 ∘ C until used. The liver samples were immediately cut into small pieces and frozen in liquid nitrogen until used. The collected serum and liver samples were used for biochemistry and histopathology studies as described previously [25].
2.6. Histopathology. Rat liver samples were fixed in 4% formalin and subjected to standard histological procedures and paraffin embedding. Liver sections (5 m in thickness) were stained with hematoxylin and eosin and evaluated for histological lesions.

Statistical Analysis.
All values were expressed as mean ± standard deviation (SD). One-way analysis of variance followed by Dunnett's multiple comparison post hoc test was used for statistical analysis of data using GraphPad Prism 6.01 (GraphPad Software Inc., San Diego, CA). A value of < 0.05 was considered to be statistically significant.

Swertianlarin Attenuates Liver Injury in Common Bile
Duct-Ligated Rats. Figure 1(a) shows that serum ALT, AST, and ALP were significantly increased in both BDL rats treated with and those without swertianlarin for 3, 7, and 14 days, compared to sham group with saline ( < 0.05, Figure 1  However, serum ALT and AST levels for 14 d were markedly decreased in the BDL rats with swertianlarin (54% and 52% of saline BDL group, resp.; < 0.05). However, the levels of these two markers were not significantly different at the earlier time points of 3 and 7 d, compared to the BDL rats without swertianlarin (Figure 1(a)). Furthermore, the serum ALP levels for 3, 7, and 14 d were not significantly different in both BDL with and without swertianlarin groups at any time (Figure 1(a)). The reduced liver injury in BDL rats with swertianlarin for 14 d was further confirmed by liver histopathology (Figure 1(c)). Moreover, the serum TBIL, DBIL, and TBA levels were noticeably increased in all BDL groups. There were no significant differences in TBIL, DBIL, and TBA between BDL groups with or without swertianlarin ( Figure 1(b)). Nevertheless, the serum levels of TBIL and DBIL tended to be lower in the BDL group with swertianlarin compared to the BDL group without swertianlarin for 14 d (Figure 1(b)). These results indicated that swertianlarin treatment decreased liver injury, whereas the serum TBIL and TBA levels in BDL rats remained unaltered for 14 d.

Swertianlarin Has Anti-Inflammatory Effect on BDL Rats.
To investigate whether swertianlarin has anti-inflammatory effects, the concentration of serum proinflammatory cytokines TNF , IL-1 , and IL-6 were measured by ELISA assay in the sham operated group and BDL groups with or without swertianlarin. The results showed that the serum TNF levels for 3 and 7 d were dramatically increased while they were not significantly increased for 14 d in cholestatic rats without swertianlarin compared to sham operated rats (Figure 2(a)). However, the levels of serum TNF in cholestatic rats with BDL for 3 and 7 d were significantly lower with swertianlarin treatment, 63% and 64% of saline BDL group, respectively ( < 0.05). However, they were not significantly different for 14 d, compared to the BDL group without swertianlarin (Figure 2(a)). Furthermore, serum IL-1 levels were significantly increased for 14 d while they were unchanged for 3 and 7 d in the BDL group with swertianlarin compared to those of the sham operated group (Figure 2(b)). However, the serum IL-1 levels for 3 and 14 d were noticeably lower in the swertianlarin BDL group (43% and 42% of BDL group without swertianlarin, resp.; < 0.05). Moreover, the serum IL-1 levels were not altered by swertianlarin for 7 d (Figure 2(b)). Figure 2(c) shows that serum IL-6 levels were increased for 14 d but was decreased for 3 d ( < 0.05) and was not different for 7 d in BDL group without swertianlarin compared to the sham operated group (Figure 3(c)). Serum IL-6 in the swertianlarin BDL group for 14 d was 61% of saline BDL group ( < 0.05), whereas the serum IL-6 levels were not affected by swertianlarin for 3 and 7 d (Figure 3(c)). These results demonstrated that swertianlarin treatment reduced levels of serum proinflammatory cytokines TNF , IL-1 , and IL-6 in this rat model.

Swertianlarin Alters the Concentration of Serum Bile Salts in Rats with Bile Duct
Ligation. The accumulation of bile salts is the major factor resulting in the liver injury in cholestasis [1,3,21]. Because an apparent decrease in liver injury in cholestatic rats was associated with swertianlarin treatment, it was determined if swertianlarin alters the concentration of serum bile salts in cholestasis. The results showed that CDCA was lower in BDL rats without swertianlarin for 3, 7, and 14 days compared with sham operated rats ( = 0.31, < 0.05, and < 0.05, Figure 3(a)). In contrast, the TCDCA levels were increased in the BDL rats without swertianlarin for 7 d but were unaltered after 3 and 14 d (Figure 3(a)). The administration of swertianlarin decreased CDCA levels after 7 and 14 d and reduced TCDCA levels after 7 d in BDL rats ( < 0.05, Figure 3(a)). The concentration of serum CA was unchanged in BDL rats without swertianlarin, compared to the sham operated group. However, the serum CA levels were decreased in BDL rats with swertianlarin for 3 ( < 0.05), 7, and 14 d ( < 0.05, Figure 3(b)). Moreover, TCA levels were only decreased in BDL rats without swertianlarin after 3 and 7 d ( < 0.05, Figure 3(b)), but not significantly changed after 14 d. However, serum TCA was significantly lower in BDL rats with swertianlarin after 3 and 14 d ( < 0.05, Figure 3(b)), but not after 7 d, when compared to BDL rats without swertianlarin. Figure 3(c) (left) shows that serum DCA levels were similar in BDL rats without swertianlarin, compared with sham group. However, the serum DCA levels were decreased in BDL rats with swertianlarin for 3, 7, and 14 d ( < 0.05 for all, Figure 3(c) left). In contrast, serum TDCA levels were increased in BDL rats without swertianlarin for 14 d but were not elevated with swertianlarin treatment ( < 0.05, Figure 3(c) right). There were no difference in TDCA levels in BDL rats with or without swertianlarin after 3 d, whereas TDCA levels were lower in BDL rats with swertianlarin after 7 d, compared with BDL rats without swertianlarin ( < 0.05, Figure 3(c)). Figure 3(d) showed that TUDCA levels were lower in cholestatic rats without swertianlarin after 3 and 7 d ( < 0.05) but were not different after 14 days. Administration of swertianlarin decreased TUDCA levels in BDL rats after 14 d ( < 0.05, Figure 3(d)) compared to BDL rats without swertianlarin. Furthermore, serum T / MCA levels were nonsignificantly different between the sham group and BDL groups without swertianlarin for 3, 7, and 14 d but were decreased in the BDL group with swertianlarin after 7 and 14 d, compared to the BDL group without swertianlarin ( < 0.05, Figure 3(e)). group after 3 and 7 d but were higher in the BDL group without swertianlarin compared to the sham group for 14 d ( < 0.05). However, serum MCA levels were decreased in the BDL group with swertianlarin for 3, 7, and 14 d ( < 0.05), compared with the BDL group without swertianlarin (Figure 3(f)). Serum MCA levels were lower in the BDL group without swertianlarin for 3 d ( < 0.05) while they were not significantly changed for 7 d and 14 d, compared with the sham group. Administration of swertianlarin for 14 d significantly decreased serum MCA levels in the BDL rats ( < 0.05, Figure 3(f), right). These results indicated that swertianlarin administration was associated with lower levels of conjugated and unconjugated bile salts in BDL rats.

Swertianlarin Does Not Affect Lipids Levels in BDL Rats.
Serum Tgh, TG, and LDL-C levels for 3 and 7 d were higher, but unchanged for 14 d in BDL saline rats, compared with the sham group ( < 0.01, Figures 4(a), 4(b), and 4(d)). However, the HDL-C levels were increased for 3 d but decreased for 14 d in BDL rats (Figure 4(c)). Furthermore, there was no difference in serum HDL-C in BDL rats after 7 d (Figure 4(c)). The results showed that levels of Tgh, TG, HDL-C, and LDL-C were not significantly different in BDL rats with or without swertianlarin ( Figure 4). These results suggest that lipid metabolism was not affected by swertianlarin in this model.

Discussion
Persisting cholestasis caused by bile duct obstruction (i.e., gallstones and pancreas tumors), hepatitis, and drugs can lead to liver failure, fibrosis, and cirrhosis [1][2][3][4][5][6]. The accumulation of bile acids in hepatocytes and hepatic chronic inflammation plays key roles in cholestatic liver injury [1][2][3]. The drugs that enhance elimination of toxic bile acids, such as UDCA and INT747 are beneficial for cholestatic patients [7][8][9][10][11][12][13][14]. In the present study, it was observed that swertianlarin attenuates liver injury and inflammation and is associated with lower concentrations of bile salts in BDL rats. Models of cholestasis can be produced by administration of some drugs, for example, -naphthylisothiocyanate (ANIT) (intrahepatic cholestasis), or by bile duct ligation (extrahepatic cholestasis) [27][28][29]. The presence of toxic bile acids is the major factor resulting in liver injury in cholestasis [1,2]. The present study showed that certain conjugated and unconjugated bile acids in serum were reduced in BDL rats by swertianlarin treatment for 3, 7, and 14 d, implying that swertianlarin attenuates cholestasis. Serum bile acid levels were reduced in BDL rats treated with swertianlarin for 14 d. These data may explain why levels of serum markers of liver injury, ALT, and AST, as well as liver necrosis determined by histopathology, were significantly decreased in BDL rats treated with swertianlarin for 14 d. Many studies have demonstrated that inhibition of bile acid synthetic enzymes (e.g., Cyp7a1), upregulation of detoxification enzymes (e.g., Cyp3a11 and Ugt2b), and bile acids transporters (e.g., Mrp3 and Mrp4) contribute to the repression of bile acid synthesis, the reduction of bile salt toxicity. This may occur by increasing water solubility and elimination of bile acids, alleviating cholestasis and liver injury in cholestatic animal models and in human cholestatic patients using UDCA and INT747 [1,2,25]. The differences in timing of alterations of serum bile acids levels in BDL rats may be due to changes in hepatic bile acid transporters, synthetic and detoxification enzymes regulated by swertianlarin. However, this hypothesis needs to be tested by more in-depth studies in future. Moreover, it was found that the lipid levels Tgh, TG, HDL-C, and LDL-C were not different in swertianlarin-treated BDL rats compared to BDL rats without swertianlarin. However, serum lipid levels were severely disturbed in all of BDL rats. These results suggested that BDL not only affected bile acid homeostasis, but also interfered with lipid homeostasis. In addition, one recent study also reported that swertianlarin from Enicostemma axillare had antioxidant and hepatoprotective effects against D-galactosamine-induced acute liver damage in rats [19]. These support the protective role of swertianlarin on cholestasis.
We also demonstrated that elevated serum levels of proinflammatory cytokines TNF for 3 and 7 d, IL-1 , and IL-6 for 14 d in BDL rats were significantly reduced by swertianlarin treatment, implying that swertianlarin has an anti-inflammatory effect on cholestatic process. These observations in BDL rats with swertianlarin treatment are similar to those previously described in adjuvant-induced arthritis and IL-1 -induced rat fibroblast-like synoviocyte models [20,21]. A recent study reported that inflammation plays an important role in the progression of the pathology of cholestasis [3]. Knockout of TNF in a cholestatic mouse model reduced liver injury and fibrosis [30]. Therefore, swertianlarin may also alter the progression of cholestasis by inhibiting inflammation. However, whether swertianlarin exerts an anti-inflammatory effect on cholestasis by the activation of NF-B and JAK signaling, as reported previously [20], remains to be clarified.
Although the swertianlarin-treated BDL rats had less liver injury, inflammation, and cholestasis than those without swertianlarin, these results are from one cholestasis model. Whether swertianlarin also exerts the protective role in other cholestatic models (e.g., lipopolysaccharide-induced cholestasis) or human cholestasis (e.g., primary biliary cirrhosis) needs further studies. Moreover, the molecular mechanism by which swertianlarin affects cholestasis remains to be determined.

Conclusions
The results demonstrated that swertianlarin from Swertia mussotii Franch attenuates liver injury, inflammation, and cholestasis in BDL rats. The findings of the present study contribute to understanding the protective role of swertianlarin in cholestasis and provide preliminary data for the development of potential drugs for the treatment of cholestasis.