Tender coconut water suppresses hepatic inflammation by activating AKT and JNK signaling pathways in an in vitro model of sepsis.

Abstract

Tender coconut water (TCW) is a natural plant product rich in phytochemicals and protects against toxic liver injury. However, the mechanism by which TCW inhibits inflammation and tissue damage is unknown. We examined the effect of TCW on primary rat hepatocyte viability, cytokine-induced gene expression and proinflammatory signaling in an in vitro model of sepsis. We observed that TCW improved hepatocyte viability and protected hepatocytes against cytokine-mediated cell death. TCW suppressed IL-1β-mediated increases in Nos2, Tnf, and Il6 mRNA and increased heme oxygenase 1 (HMOX1) protein. TCW inhibited iNOS expression through activation of AKT and JNK pathways since inhibition of PI3K and JNK signaling reduced TCW’s effect on iNOS protein expression and activity. These results demonstrate that TCW reduces proinflammatory gene expression and hepatocyte injury produced by elevated inflammatory cytokines and nitric oxide production.

Graphic abstract:

Tender coconut water (TCW) reduces the generation of NO induced by IL-1β in isolated primary rat hepatocytes by activating JNK and AKT signaling pathways and inhibits Tnf and Il6 production. TCW improved hepatocyte viability and enhanced expression of the antioxidant HMOX1.

1. Introduction

Nitric Oxide (NO) has diverse physiological and pathophysiological roles (Tuteja, Chandra, Tuteja, & Misra, 2004). NO produced by the constitutively active neuronal NOS (N-NOS, NOS1) and endothelial NOS (E-NOS, NOS3) regulates neurotransmission, smooth muscle function, and blood vessel contraction (Bredt & Snyder, 1992; Buchwalow et al., 2002). NO produced by the inducible NOS (iNOS, NOS2) plays an essential role in host defense against infection but excessive NO production by iNOS causes tissue damage, chronic inflammation and can contribute to liver fibrosis, transplantation rejection and organ dysfunction (Nussler & Billiar, 1993; Cannon, 1999). NO is increased in the liver of patients in septic shock and in animal models of shock, sepsis, and liver transplantation due to ischemia/reperfusion (I/R) injury and proinflammatory cytokine production (Hierholzer et al., 1998; Shah & Kamath, 2003). Chronic liver inflammation increases the risk of hepatocellular carcinoma (HCC) and elevated iNOS has been implicated in the development of HCC (Kawanishi, Hiraku, Pinlaor, & Ma, 2006). Hepatocyte iNOS is upregulated by cytokines and we have shown that cAMP, glucagon and insulin downregulate cytokine-mediated iNOS expression (Harbrecht, Taylor, Xu, Ramalakahmi, Ganster & Geller, 2001; Zhang, Li, & Harbrecht, 2011; Zhang, Perpetua, Fulmer, & Harbrecht, 2004). Decreasing NO production in the liver or scavenging excess NO during shock, I/R, inflammation and transplantation decreases liver injury and tissue damage (Chen, Liao, Yang, & Wang, 2014; da Cunha, Lopes, Panis, Cecchini, Pinge-Filho, & Martins-Pinge, 2017) suggesting that reducing NO production from iNOS can be beneficial and protect the liver during these insults.

Tender coconut water (TCW) is the nutrient rich liquid endosperm of young (6–8 mos.) coconuts (Cocos nucifera L) that contains bioactive compounds including vitamins, minerals, sugars, amino acids, peptides, proteins, plant metabolites, and phytohormones (Supplementary Table 1) and is commonly consumed in tropical countries (Adams & Bratt, 1992; DebMandal & Mandal, 2011). Its composition makes it a useful rehydrating fluid and it has been used to treat diarrhea, dysentery, fever and body aches (Adams & Bratt, 1992; DebMandal & Mandal, 2011). TCW has anti-diabetic, anti-inflammatory, anti-thrombotic, antibacterial, wound healing and anti-cancer activities (Bhagya, Prema, & Rajamohan, 2012; Loki & Rajamohan, 2003; Prabhu et al., 2014; Preetha, Devi, & Rajamohan, 2015; Rao & Najam, 2016).

Several bioactive natural products are sources of compounds that have been used either prophylactically or therapeutically to prevent/alleviate diseases. The advantage of using natural products is that they are usually well-tolerated with minimal side effects. Many flavonoids, coumarins, and tannins found naturally in plants suppress NO generation in experimental models, suggesting that natural products may inhibit inflammatory pathways including iNOS (Murakami, 2009). Although TCW reduces paw edema and accelerates wound healing in experimental models, the mechanism behind its anti-inflammatory properties has not been determined (Radenahmad et al., 2012; Rao & Najam, 2016). Given TCW’s reported anti-inflammatory effects, we hypothesized that TCW would regulate the expression of inflammatory mediators and cytokine-mediated NO production, Nos2 mRNA and iNOS protein expression in primary rat hepatocytes.

2. Materials and methods

2.1. Materials

Fresh TCW was collected from coconuts of Maphora Nam Hom variety (Cocos nucifera. L. var. nana) between 6–8 months of maturity purchased at a local grocery store (the coconuts were from Thailand and certified to be organic by the USDA). The TCW from 2–4 coconuts was pooled, sterile filtered and stored at 4°C in 50 mL aliquots. Commercial coconut water products marketed under different brands were also purchased from a local grocery and sterile filtered as above (Supplementary Table 2, & 3). Williams Medium E, penicillin, streptomycin, L-glutamine and HEPES were from Life Technologies (Carlsbad, CA). Insulin was from Lilly (Indianapolis, IN). The polyclonal antibody to iNOS (catalogue number 610431) was from BD Bioscience (Billerica, MA), HMOX1 (catalogue number PC340) was from Calbiochem (San Diego, CA) and β-Actin (catalogue number MAB1501) was from Millipore (Burlington, MA). Antibodies to total-GSK3α/β (catalogue number 5676), GSK3α/β phosphorylated at S21/S9 (catalogue number 9331), total-AKT (catalogue number 9272), AKT phosphorylated at S473 (catalogue number 5012), total-JNK1/2 (catalogue number 3708), phopsho-JNK1/2 (catalogue number 4668), total-c-Jun (catalogue number 9164), c-Jun phosphorylated at S63 (catalogue number 2361), and GAPDH (catalogue number 5174) were purchased from Cell Signaling Technology (Danvers, MA). LY294002 and SP600125 were from Calbiochem (San Diego, CA), and human recombinant interleukin 1β (IL-1β) from Dupont (Boston, MA). All other chemicals used in this study were reagent grade and of the highest purity available.

2.2. Primary hepatocyte isolation and culture

Primary hepatocytes were isolated from male Sprague-Dawley rats (150–200 g; Envigo, Indianapolis, IN, USA) using a modified collagenase perfusion technique (Zhang, Li & Harbrecht, 2011). Experimental animals were cared for according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) following the guidelines prescribed by the National Institutes of Health’s Guidelines for the Care and Use of Laboratory Animals. The isolated and purified hepatocytes (98% pure with >95% viability by trypan blue exclusion) were cultured on collagen-coated plates in Williams Medium E with L-arginine (0.5 mM), L-glutamine (2 mM), HEPES (15 mM), penicillin, streptomycin and 10% charcoal-stripped FBS (CS-FBS; HyClone Laboratories, Logan, UT) for 4 hours. The unattached cells were washed away and the hepatocytes were incubated with insulin-free media with 5% CS-FBS since insulin is known to suppress cytokine mediated iNOS expression in cultured rat hepatocytes (Harbrecht, Nweze, Smith, & Zhang, 2012). Media containing varying concentrations of TCW was prepared by adding TCW (v/v) and sterile water to 2 X William E containing 10% CS-FBS to ensure that the media and FBS were not diluted by the addition of TCW. The hepatocytes were pretreated with the indicated amount of TCW in media prepared as described above for 30 min before the addition of 200 U/ml of IL-1β. In experiments involving LY294002 or SP600125, control hepatocytes and those treated with IL-1β alone received 0.001% DMSO in the culture media as vehicle control, equivalent to the amount present in hepatocytes treated with the inhibitors. Hepatocytes isolated from 3 different rats were used under identical experimental conditions to generate three independent data sets (biological replicates).

2.3. Nitrite (NO₂⁻) measurement

NO₂⁻ from hepatocyte culture supernatants was measured as an index of NO production by the Griess reaction (Harbrecht et al., 2001).

2.4. Western blot

Proteins (50 μg) from primary hepatocytes were resolved on 12% SDS-PAGE gels and transferred to nitrocellulose membranes for Western blot analysis as described (Lakshmanan, Zhang, Nweze, Du, & Harbrecht, 2015). Membranes were first probed for the phosphorylated form of the protein, then stripped and re-probed for total levels of the respective protein using the appropriate antibody before probing for internal standard, β-actin/GAPDH. Quantification of band intensity was performed using ImageJ software (Lakshmanan et al., 2015).

2.5. RNA extraction and Q-PCR

TRIzol Reagent (Life Technologies) was used to isolate total RNA from the hepatocytes according to the manufacturer’s protocol. TaqMan Reverse Transcription reagent (Applied Biosystems) was used to generate cDNA from total RNA. The proprietary probes for rat Nos2 (Rn00561646_m1), Serpine1 (Rn01481341_m1), Tnf (Rn99999017_m1), Il6 (Rn00561420_m1) and Gapdh (Rn01775763_g1) for Q-PCR were purchased from Life Technologies (Grand Island, NY). Samples were analyzed using a StepOne Plus PCR machine (Applied Biosystems) in duplicates for each set of experiment, and the average values were used for quantification. Gapdh was used as the endogenous control. The comparative C_T method (cycle threshold) was used for quantification of gene expression.

2.6. MTT assay

To measure hepatocyte viability, MTT assay was employed as described (Mosmann, 1983).

2.7. Fractionation of TCW

TCW was acidified to pH 3.0 with acetic acid, filtered. Total acidified TCW sample (80 ml) was divided into two portions. One portion (16 ml) was stored at −80 °C as unfractionated control. The remaining 64 ml was subjected to reverse phase gravity flow C18 SPE column (Bond Elute C18; 500 mg; 3 mL, Agilent Technologies, Santa Clara, CA) that was equilibrated sequentially with methanol–acetic acid (100:1, v/v), methanol–water–acetic acid (50:50:1, v/v/v), methanol–water–acetic acid (30:70:1, v/v/v), and then finally with water as previously described (Ge et al., 2005). The column was washed with 4 mL of water adjusted to pH 3.0 with acetic acid. The bound fraction was then eluted with 6 mL of ethanol-water-acetic acid (80:20:1, v/v/v) and the solvent evaporated under vacuum with the final product dissolved in 50% ethanol. The fractions and the starting material were neutralized to pH 7.4 by titrating with NaOH to test the ability of various fractions to suppress IL-1β-induced NO₂⁻ production by primary rat hepatocytes. The flow through, wash and eluent were collected and stored at −80 °C. For HPLC analysis of the fractions, the samples were freeze-dried and dissolved in water at 5 mg dry mass/ml. Total phenolic content was determined as described (Slinkard & Singleton, 1977) and modified (Siriwoharn, Wrolstad, Finn, & Pereira, 2004; Tsao & Yang, 2003). Based on phenolics content estimates, each sample was diluted to 0.1 mg phenolic (gallic acid equivalent)/ml water and subjected to HPLC analysis. HPLC analysis was performed using an Agilent 1100 system equipped with a degasser, quaternary pump, automated injector, column oven and diode-array detector (set to 280 nm with a bandwidth of 16 nm, reference at 360 nm with bandwidth of 100 nm, slit at 2 nm, peak width [response time] at 0.4min [8s]). The system was fitted with a 250 ×4.6 mm Altima HP 5 μm C18-AQ column. The flow rate was set to 1.5 ml/min solvent A (1% acetic acid in water) and 0.02 ml sample was injected. Samples were eluted as follows: 100% A for 5 min increasing linearly to 50% solvent B (1% acetic acid in methanol) from 5 to 20 min then increasing linearly to 100% solvent B from 20 to 25 min, holding at 100% B until 30 min then returning to 100% solvent A from 30 to 35 min.

2.8. Statistical analysis

The results are expressed as the mean ± SD of 3 independent experiments using hepatocytes isolated from at least 3 different rats. Data were analyzed by one-way analysis of variance (oneway ANOVA) followed by post hoc Tukey test for p value determination. Data involving multiple components were analyzed for significance using 2-way ANOVA, and if significance between groups was evident, Tukey’s post hoc was used for pairwise comparisons and p values. A value of p < 0.05 was considered statistically significant. Statistical analysis was performed using the IBM SPSS 21 software.

3. Results

3.1. TCW enhances primary rat hepatocyte survival in vitro

We measured the effect of TCW on the viability of cultured primary rat hepatocytes in the absence and presence of IL-1β using the MTT assay. Without TCW supplementation, cytokine treatment decreased hepatocyte viability slightly at 24 h compared to un-treated cells (Fig. 1a). TCW supplemented hepatocytes showed a concentration-dependent increase in viability, even in the presence of IL-1β. The effect was highest with 10% and 25% TCW supplementation. Furthermore, primary rat hepatocyte cultures supplemented with TCW maintained their typical morphology and phenotype in vitro for at least 5 days without any additional supplemental growth factors other than 5% CS-FBS (Fig. 1b). In contrast, primary rat hepatocytes cultured in media with 5% CS-FBS alone lost their typical appearance and morphologic evidence of apoptosis (small rounded cells, Fig. 1b, top panel) was apparent.

Fig. 1.

TCW enhances the viability of primary rat hepatocytes. (a) The viability of rat primary hepatocytes receiving different concentrations of TCW was assessed by MTT assay after 24 h of treatment. The bars represent the mean ± SD, n = 3. *p < 0.05 vs control; #p < 0.05 vs IL-1β treatment; ^&p < 0.05 vs 1% and 5% TCW treatment and ^$p < 0.05 vs 1% and 5% TCW + IL-1β treatment (b) The morphology of primary rat hepatocytes grown in the presence or absence of 10% TCW supplementation for 5 days is shown. The cells were grown in medium as described in Methods. C, Control; TCW, Tender Coconut Water.

3.2. TCW inhibits IL-1β-mediated NO production by primary rat hepatocytes

To determine if TCW has anti-inflammatory properties in hepatocytes, primary rat hepatocytes were treated with 200 U/mL IL-1β for 20 h to induce NO₂⁻ (Fig. 2a). Pretreatment (30 min) with TCW inhibited IL-1β-induced NO₂⁻ in a concentration-dependent manner. TCW also abrogated the IL-1β-mediated increase in iNOS protein expression (Fig. 2b) and IL-1β-induced Nos2 transcript expression (Fig. 2c). Boiling TCW for 10 min did not diminish its ability to inhibit IL-1β mediated iNOS protein expression or suppress NO₂⁻ production (data not shown) demonstrating that the active ingredient(s) of TCW was heat-stable. These data demonstrate that TCW inhibits IL-1β- induced Nos2, iNOS protein, and NO₂⁻ production in primary rat hepatocytes. We compared commercial coconut water products marketed under different brands to fresh TCW. Commercially available coconut water was equally as effective at inhibiting IL-1β mediated NO₂⁻ production by rat primary hepatocytes as freshly prepared TCW (Table 1).

Fig. 2.

TCW suppresses NO₂⁻ production, iNOS protein and iNos mRNA expression in primary rat hepatocytes. (a) Cells were pretreated with the indicated concentration (volume percent) of TCW in William E medium for 30 min. The cells were then treated with 200 U/mL of IL-1β for 20 h. Levels of NO₂⁻ in the media were determined by Griess method and compared to NO₂⁻ levels in cells that were treated with IL-1β alone. The cells in the control group did not receive either TCW or IL-1β. (b) A representative Western-blot for the effect of TCW concentration on iNOS protein expression with the lower panel showing the densitometry quantitation of iNOS/β-actin with the IL-1β-induced value set to 100% for comparison. (c) Nos2 mRNA levels in rat primary hepatocytes were measured 180 min after IL-1β (200 U/mL) treatment and compared to hepatocytes receiving no IL-1β (control), pre-treated with TCW (10%) alone, and TCW + IL-1β. Gapdh expression was used as the internal control to normalize iNOS expression levels. The values represent the mean ± SD; n = 4 for A, and n = 3 for B and C. *p < 0.005 vs control; ^#p < 0.05 vs IL-1β; ^##p < 0.005 vs IL-1β; ^$p < 0.05 vs 2.5% TCW + IL-1β; ^&p < 0.05 vs 5% TCW+ IL-1β and ^@p < 0.05 vs 10% TCW + IL-1β. C, Control; IL, IL1β, TCW, Tender Coconut Water. Supplementary Fig. 3 show full blots of westerns in Fig. 2b.

Table 1.

Comparison of fresh TCW with commercial coconut water (marketed under different brands, A and B) for their ability to inhibit IL-1β-mediated nitrite production by rat primary hepatocytes. Primary rat hepatocytes were pretreated with 10% TCW or coconut waters A or B for 20 min. prior to addition of 200 U/ml IL-1β. Nitrate levels were measured by the Griess reaction after 24 h.

Treatment	Relative Nitrite Levels (% control ± SD)
Media alone	8.15 ± 0.55
Media + IL-1β	100.00 ± 0.00
10% Fresh TCW + IL-1β	34.20 ± 8.78*
10% Brand A TCW + IL-1β	31.66 ± 1.33*
10% Brand B TCW + IL-1β	26.78 ± 6.32*

^*p < 0.05 when compared to hepatocytes receiving IL-1β alone.

3.3. TCW alters the expression of inflammatory mediators in primary rat hepatocytes

To determine whether the anti-inflammatory effect of TCW was specific to iNOS, we measured the mRNA expression of other inflammatory hepatocyte genes such as the acute phase protein Serpine1 (PAI-1), Tnf (TNFα), and Il6 (IL6). TCW treatment alone increased hepatocyte Serpine1 mRNA levels, comparable to that of IL-1β-treated hepatocytes (Fig. 3a). The combination of TCW and IL-1β-treatment further increased Serpine1 mRNA levels but the increase was not statistically significant when compared to hepatocytes treated with TCW or IL-1β alone (Fig. 3b). IL-1β increased Tnf mRNA levels and TCW showed a small but statistically significant decrease in IL-1β-stimulated Tnf expression (Fig. 3b). IL-1β did not significantly increase Il6 mRNA levels in hepatocytes while TCW reduced basal Il6 transcript levels but not when cultured with IL-1β (Fig. 3c).

Fig. 3.

Effect of TCW on Serpine1, Tnf, Il6 and HMOX1 expression in primary rat hepatocytes. Primary rat hepatocytes were pre-incubated without or with 10% TCW before the addition of IL-1β (200 U/mL). After 180 min, total RNA was isolated and Serpine1 (a), Tnf (b) and Il6 (c) mRNA levels measured by Q-PCR using Gapdh as the internal control. (d) Protein extracts (50 μg) from hepatocytes treated with TCW and incubated over-night with IL-1β (200 U/ml) were analyzed for HMOX1 levels by western blot. The blot was stripped and re-probed for β-actin as a loading control. The lower panel shows the summary of densitometry analysis of HMOX1 protein in three replicate westerns. The HMOX1/β-actin in the IL-1β-induced sample was set to 1 for comparison. In all experiments, hepatocytes that received media alone served as control. The bars represent the mean ± SD, n = 3. *p < 0.05 and **p < 0.001 vs control; ^#p <0.05 and ^##p < 0.01 vs IL-1β; ^$p < 0.005 vs TCW. C, Control; IL, IL-1β; TCW, Tender Coconut Water. Supplementary Fig. 4 show full blots of westerns in Fig. 3d.

Heme oxygenase-1 (HMOX1) is an antioxidant and is cytoprotective against oxidative stress (Waza, Hamid, Ali, Bhat, & Bhat, 2018) but we found that IL-1β was a weak inducer of HMOX1 in cultured hepatocytes (Figure 3d). However, TCW markedly increased the expression of HMOX1 protein in primary rat hepatocytes and the addition of IL-1β to TCW-treated cells caused a further increase in the HMOX1 protein (Fig. 3d). These results support the anti-inflammatory properties of TCW and demonstrate that TCW increases the expression of anti-inflammatory HMOX1 protein in primary rat hepatocytes.

3.4. TCW activates AKT signaling in primary hepatocytes

We previously demonstrated that AKT regulates cytokine-stimulated hepatic iNOS expression (Harbrecht et al., 2012; Zhang et al., 2011). Thus, we examined the impact of TCW (10%, v/v) on AKT signaling in primary rat hepatocytes. As previously reported (Harbrecht et al., 2012; Zhang et al., 2011), IL-1β increased AKT phosphorylation in hepatocytes and this stimulation was blocked by LY294002, a specific inhibitor of PI3K/AKT (Fig. 4a). TCW alone significantly increased AKT phosphorylation (18-fold increase) (Fig. 4a). Interestingly, we observed that the increase in p-AKT/AKT was greater at 30 than 60 min after TCW treatment. IL-1β, when combined with TCW, increased AKT phosphorylation to levels that were significantly greater than that stimulated by IL-1β or TCW alone (Fig. 4a). LY294002 caused a significant reduction in TCW-mediated AKT phosphorylation in both the presence and absence of IL-1β (Fig. 4a). Similar to our earlier reports (Lakshmanan et al., 2015; Zhang et al., 2011), the LY294002 -induced inhibition of AKT phosphorylation was associated with a significant increase in IL-1β-mediated NO₂⁻ production and iNOS protein expression (Fig. 4b and 4c). LY294002 partially reversed the inhibitory effects of TCW on the IL-1β-mediated nitrite production (Fig. 4b) and iNOS protein expression (Fig. 4c). Together, these data suggest that TCW mediates its suppression of iNOS protein expression in part through activation of AKT phosphorylation.

Fig. 4.

TCW activates AKT signaling in primary rat hepatocytes. (a) A representative Western blot on the effect of TCW (10%), IL-1β (200 U/mL) and LY294002 (10μM) on phosphorylation of AKT at 30 and 60 min after IL-1β stimulation. The lower panel shows the quantitative densitometric analysis. (b) and (c) show the effect of TCW and LY294002 on IL-1β simulated NO₂⁻ and iNOS protein levels, respectively. The lower panel in c shows the quantification of iNOS protein normalized to β-actin, the loading control, in the same lane, as a loading control. (d) The effect of TCW and LY294002 on GSK3 phosphorylation. The lower paned in (d) shows the densitometric analysis for GSK3 level. GAPDH was used as the internal standard in (a) and (d). The bars in (a),(b), (c) and (d) represent the mean ± SD, n = 3. *p <0.05, **p < 0.005 vs control; ^#p < 0.05 vs IL-1β; ^€p < 0.05 vs TCW; ^&p < 0.005 vs TCW; ^$p < 0.01 vs TCW+IL-1β; ^@p < 0.05 vs LY; ^©p < 0.05 vs LY + TCW and ^£p < 0.05 vs LY+IL-1β. C, Control; TCW, Tender Coconut Water; IL, IL-1β; LY, LY294002. Supplementary Fig. 5–7 show full blots of westerns in this Figure.

GSK3 is a downstream target of AKT and is a positive regulator of IL-1β-induced iNOS expression in hepatocytes (Lakshmanan et al., 2015). Phosphorylation of GSK3 by AKT leads to its inactivation. TCW increased the phosphorylation of GSK3α/β (Fig. 4d) and the phosphorylation pattern was parallel to that of phospho-AKT (Fig. 4a), including a larger effect at 30 than 60 min. Preincubation with LY294002 prior to TCW treatment decreased phospho-GSK3α/β levels (Fig. 4d). These results demonstrate that TCW activation of PI3K/AKT leads to GSK3α/β inhibitory phosphorylation in primary rat hepatocytes.

3.5. TCW activates JNK signaling in primary rat hepatocytes

JNK signaling inhibits cytokine-mediated hepatocyte iNOS expression (Zhang, Perpetua, Fulmer, & Harbrecht, 2004). We therefore evaluated the effect of TCW on JNK signaling to determine if this pathway contributes to the inhibitory effect of TCW on IL-1β-induced iNOS expression. As previously shown, IL-1β increased JNK phosphorylation (Fig. 5a) (Zhang et al., 2004). TCW alone induced JNK1/2 phosphorylation to a level significantly greater than that of IL-1β alone at both 30 and 60 min (Fig. 5a). Although not statistically significant at 30 min, the combination of IL-1β with TCW showed a trend of increasing JNK phosphorylation beyond that of TCW alone and this difference was more apparent at 60 min (Fig. 5a).

Fig. 5.

TCW activates JNK signaling pathway in rat primary hepatocytes. (a) Primary rat hepatocytes were treated with TCW (10%) and IL-1β (200 U/mL) for the indicated times and analyzed for phospho- and total-JNK1/2 protein 30 and 60 min after the addition of IL-1β. (b) The western blot shows the effect of TCW on phospho-(S63) and total c-Jun. The lower panel in (a) and (b) shows the result of the densitometric analysis with GAPDH as the loading control. (c) The effect of JNK inhibitor, SP600125 (10μM), on the TCW mediated effects on IL-1β mediated NO₂⁻ generation and (d) iNOS protein expression in rat primary hepatocytes. The lower panel in (d) is the densitometry data on the iNOS band intensity normalized to β-actin as a loading control. The bars represent the mean ± SD, n = 3 in (a), (b) and (d); and n = 4 in (c). *p < 0.05 vs control; **p < 0.005 vs control; ^#p < 0.005 vs IL-1β; ^$p < 0.05 vs IL-1β; ^€p < 0.05 vs TCW; ^@p <0.05 vs SP+ IL-1β; ^£p > 0.05 vs SP+ IL-1β. C, Control; IL, TCW, Tender Coconut Water; IL-1β; SP, SP600125. Supplementary Fig. 8–10 show full blots of westerns in this Figure.

In hepatocytes, JNK phosphorylation leads to increased c-Jun, which suppresses iNOS (Zhang et al., 2004). Hence, we measured phospho-c-Jun (Ser63) levels in hepatocytes treated with 10% TCW. TCW increased phosphorylation of c-Jun (Fig. 5b), paralleling the increase in phospho-JNK (Fig. 5b). The JNK inhibitor SP600125 was used to block JNK signaling and, when cultured with hepatocytes alone, SP600125 did not alter control NO₂⁻ production or iNOS expression. SP600125 caused a significant increase in IL-1β-mediated NO₂⁻ and iNOS protein expression comparted to IL-1β alone (Fig. 5c and d). The TCW-induced suppression in NO₂⁻ and iNOS protein in IL-1β-treated hepatocytes was partially reversed by SP6001255, suggesting that JNK regulates the TCW-mediated repression of IL-1β-induced iNOS.

3.5. Non-polar TCW components have iNOS suppressing activity in rat hepatocytes

To characterize the active ingredient(s) in TCW that are responsible for inhibiting IL-1β-induced inflammation, TCW was fractionated using a preparative C18 column and iNOS expression used to screen for biologic activity. The unbound polar fraction (flow-through) retained iNOS suppressing capacity, as evident by the reduction in IL-1β-stimulated NO₂⁻ levels in hepatocytes pre-treated with this fraction (Table 2). The fraction eluted from the C18 column lacked the iNOS inhibiting activity of TCW (Table 2). The total phenolic content of the fractions shows the enrichment of phenolics in the eluent (Supplementary Fig. 1). Further analysis of the C18 column fractions by HPLC revealed peaks found in the flow through fraction (peaks 1–4 in Supplementary Fig. 2) were not found or found at very low levels in the eluent. Conversely, peaks 5–6 that were found in the eluent fraction were not found or found at very low levels in the flow through. Thus, differences in iNOS inhibiting activity correlates with phytochemcials that are enriched in flow through and are absent in eluent (Supplementary Fig. 2).

Table 2.

Identification of active fraction of TCW after C18 column separation. The ability of the unbound and bound fractions of TCW from a C18 column were tested for their ability to inhibit IL-1β mediated nitrite production by rat primary hepatocytes (see Table 1 for experimental details).

Treatment	Relative Nitrite Levels (% control ± SD)
Media	7.70 ± 1.63
Media + IL-1β	100.0 ± 0.00
10% TCW + IL-1β	42.83 ± 9.47*
10% C18 Flow-through + IL-1β	38.39 ± 7.58*
10% C18 Eluate + IL-1β	103.73 ± 4.37

^*p < 0.05 when compared to hepatocytes receiving IL-1β alone.

4. Discussion

Decreasing the deleterious effects of inflammation is a therapeutic strategy for several chronic liver diseases including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and hepatitis C infection (Campo, Gallego, & Grande, 2018). Down-regulating inflammation and the induced iNOS expression that is associated with tissue injury following ischemia, shock, and sepsis is a potential strategy to improve clinical outcome.

Tender coconut water is a natural plant based product that has anti-oxidative properties in several experimental models and protects the liver from chemical toxin-induced injury (Bhagya et al., 2012; Loki & Rajamohan, 2003). Previous studies reported that oral administration of TCW reduced oxidative stress induced by isoproteronal-induced myocardial infarction (Prathapan & Rajamohan, 2011) and reduced inflammation (Rao & Najam, 2016) in rats. However, the mechanism for the anti-inflammatory effect of TCW is unknown. Here we report for the first time that TCW inhibits hepatocyte inflammation by reducing IL-1β-induced Tnf and Il6 transcript expression while increasing acute phase protein (Serpine1 transcript) and antioxidant (HMOX1 protein) expression in primary rat hepatocytes. TCW inhibited IL-β mediated reduction in Nos2 transcript levels and iNOS protein in primary rat hepatocytes via stimulation of AKT and JNK signaling pathways.

NO is an important immunomodulatory molecule and suppression of NO production by inhibiting iNOS to reduce inflammation has been successful in animal models (Clancy, Amin, & Abramson, 1998; Cross & Wilson, 2003; Vallance & Leiper, 2002). In the present study, we show that TCW not only suppressed nitrite production by primary hepatocytes, but also inhibited Nos2 mRNA and iNOS protein expression (Fig. 2a–c). This is consistent with studies in which TCW suppressed nitrite production after an oxidative insult (Loki & Rajamohan, 2003; Manna et al., 2014), although the mechanism of TCW-mediated suppression of iNOS was not examined in these studies. We previously demonstrated that AKT and JNK are important negative regulators of hepatic iNOS (Zhang et al., 2011, 2004). The data presented here demonstrate that TCW suppresses hepatic inflammation and Nos2 mRNA and iNOS protein expression (Fig. 2) in part by activating AKT and JNK signaling pathways (Fig, 4 & 5). The PI3K-AKT pathway mediates inflammation and inhibition of this pathway increased serum cytokine levels and decreased survival in an animal model of sepsis (Schabbauer, Tencati, Pedersen, Pawlinski, & Mackman, 2004; Williams et al., 2004). It is possible that TCW activates additional intracellular signaling pathways and we do not rule out the involvement of other signaling pathways in the TCW-mediated effect on Nos2 mRNA and protein expression. TCW suppresses NF-κB nuclear translocation following stressors such as heat-shock and hydrogen peroxide (H₂O₂) (Kumar, Manna, & Das, 2018; Manna et al., 2014) and regulation of NF-κB can potentially contribute to anti-inflammatory effects in our model. The role of TCW in suppressing cytokine and IL-1β mediated nuclear translocation of NF-κB in sepsis or infection remains to be fully determined.

Work from other laboratories has demonstrated that in vivo, TCW reduces CCl₄-and H₂O₂-induced oxidative damage as well as fructose-induced oxidative stress to rat liver (Bhagya et al., 2012; Loki & Rajamohan, 2003). In these studies, TCW upregulated the free-radical scavenging enzymes superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX), but the mechanism for these changes was not identified. Preliminary studies in our lab also show that TCW decreases liver Nos2 in vivo in a hepatic ischemia-reperfusion injury model in mice (unpublished data). In the liver, iNOS is a major contributor to free radicals produced in hypoxic and ischemic conditions such as infection and injury (Iwakiri & Kim, 2015) and our finding of decreased iNOS expression by TCW likely has an anti-oxidant effect. We demonstrate that TCW increased the protein levels of HMOX1 (Fig. 3d), an anti-oxidant and anti-inflammatory protein that is important in the hepatic response to inflammation and oxidative injury, suggesting potent anti-oxidant effects by TCW although oxidant-mediated membrane damage was not directly measured in these studies. TCW suppressed the IL-1β mediated expression of Tnf and TNFα is an established mediator of inflammation in liver (Fig. 3b). While hepatocytes may not produce as much TNFα as other cells such as macrophages, these findings suggest that TCW may favorably alter the anti-oxidant defenses and inflammatory response of hepatocytes as a component of its protective effects. These mechanisms could account for the decreased cellular death in the presence of TCW (Fig. 1a). This finding is consistent with earlier studies demonstrating TCW protects hepatocytes against H₂O₂ mediated oxidative damage and testis against heat-induced damage (Kumar et al., 2018; Manna et al., 2014). GSK3 is a negative regulator of Nrf2 activity and is essential for the production of inflammatory cytokines (Biswas et al., 2014; Jain & Jaiswal, 2007; Martin, Rehani, Jope, & Michalek, 2005; Wang, Brown, & Martin, 2011). Inhibition of GSK3 decreases hepatic cytokine production in animal models of acute liver failure and ischemia/reperfusion (Chen et al., 2012; Ren et al., 2011). Thus, it is conceivable the observed decrease in pro-inflammatory Tnf and Il6 and the increase in anti-inflammatory HMOX1 by TCW is through the AKT-mediated inhibition of GSK3. This would be consistent with the increase in inhibitory phosphorylation of GSK3 at S9/21 in the presence of TCW seen in our experiments (Fig. 4d).

The active ingredients of TCW include plant secondary metabolites, phytohormones, vitamins and amino acids. For example coumarin, 4-hydroxycoumarin, coumaric acid, ferulic acid glucoside, procyanidin, shikimic acid and quinic acid were identified in TCW and are known to exhibit antioxidative and hepatoprotective effects in experimental settings (Kumar et al., 2018; Manna et al., 2014). Our fractionation approach leads to clear separation of phytochemical constituents based on polarity (Supplementary Fig. 2; see peaks 1–4 in flow through (panel B) and 5 and 6 in eluent (panel D). Since iNOS suppressing activity was demonstrated in the flow through but not eluent fractions, the more polar constituents of the flow through fraction appear to correlate well with bioactivity and need to be further characterized in future work.

Conclusion

TCW represses hepatocyte IL-1β mediated inflammatory damage by inhibiting Nos2 mRNA and iNOS protein expression through AKT and JNK signaling pathways in vitro. Our studies show that TCW decreased hepatocyte expression of pro-inflammatory cytokines and increased expression of acute phase proteins Serpine1 and HMOX1. The results suggests that TCW could potentially be beneficial as a therapeutic agent in conditions where hepatic Nos2 expression is upregulated. Further study of the beneficial effects of TCW are merited.

Highlights.

• IL-1β-induced expression of hepatocyte iNOS is suppressed by Tender Coconut Water (TCW)

• TCW inhibits IL-1β-induced Tnf and Il6 expression

• TCW increases expression of Serpine1 mRNA and HMOX1 protein expression

• TCW activates AKT and JNK signaling pathways

Abbreviations:

AKT

Protein kinase B

CS-FBS

charcoal-stripped fetal bovine serum

HMOX1

Heme oxygenase 1

GSK3

Glycogen synthase kinase-3

HCC

hepatocellular cancer; Heamoxygenase-1

iNOS/Nos2

Inducible nitric oxide synthase

IL-1β

Interleukin-1beta

NO

Nitric oxide

Il6

Interleukin 6

I/R

Ischemia/reperfusion

JNK

c-Jun N-terminal kinase

PAI-1

Plasminogen activator inhibitor-1

PI3K

Phosphatidylinositol 3-kinase

TCW

Tender coconut water

TNFα/Tnf

Tumor necrosis factor alpha