Fucoidan from Cucumaria frondosa Inhibits Pancreatic Islets Apoptosis Through Mitochondrial Signaling Pathway in Insulin Resistant Mice

Abstract

Fucoidan from sea cucumber is reported to exhibit anti-hyperglycaemic effect, but its influence on pancreatic islets is lacking. The effects of fucoidan from Cucumaria frondosa (Cf-FUC) on inhibiting pancreatic islets apoptosis in high-fat high-sucrose diet (HFSD)-induced insulin resistant mice were investigated in this study. The results showed that Cf-FUC significantly prevented HFSD-injured pancreatic islets, decreased blood glucose, insulin and TNF-α levels, and increased adiponectin level. Cf-FUC significantly reduced Bid, Bax, cytochrome c, caspase 9, and caspase 3 mRNA expression levels, and increased Bcl-2 and Bcl-xL mRNA expression levels. Cf-FUC also caused significant down-regulation of t-Bid, Bax, cytochrome c in cytoplasm, caspase 9, and cleaved-caspase 3 proteins, and up-regulation of Bcl-2 and Bcl-xL proteins. Furthermore, Cf-FUC enhanced the effects of RSG. This study demonstrates that Cf-FUC inhibits pancreatic islets apoptosis via inhibiting mitochondrial pathway, which may provide a dietary intervention hyperglycaemia-induced pancreatic islets apoptosis.

Introduction

Diabetes mellitus type 2 is becoming increasingly common in many countries (Shaw, 2009). It is characterized by hyperglycemia in the context of relative deficiency of insulin and insulin resistance (Alberti and Zimmet, 1998; Muoio and Newgard, 2008). The inability of pancreatic β-cell to provide the body with an insufficient insulin supply to compensate for insulin resistance is an early defect in the natural history of type 2 diabetes (Moon et al., 2013). The reduction of β-cell in the pancreas mass has been reported to be a pathological hallmark in the development of type 2 diabetes (Jurgens et al., 2011; Lee et al., 2011). The β-cell mass is reduced to a variable extent in patients with type 2 diabetes (Maedler, 2008). It is also reported that increased β-cell apoptosis can lead to the continuous loss of β-cells (Meier and Bonadonna, 2013). Few of the pharmacological therapies for type 2 diabetes are reported to prevent the progressive decline in β-cell function or inhibition of its apoptosis (Hou et al., 2013). Therefore, therapeutic agents that can halt or prevent pancreatic β-cell apoptosis failure will likely have a major impact on disease progression.

Inflammation plays a critical role in the development of insulin resistance and type 2 diabetes (Kang et al., 2010; Lee, 2013). It has been reported that inflammatory mediators, including tumour necrosis factor-α (TNF-α), are involved in the pathogenesis of β-cell dysfunction of diabetes (Bouzakri et al., 2011; Cai et al., 2011; Marselli et al., 2013). Recently, a specific role has been proposed for TNF-α in impairing insulin secretion and inducing β-cell apoptosis in pancreatic cell lines (Zhang et al., 2011). Adiponectin is an insulin-sensitizing hormone which is negatively associated with insulin resistance (Blesso et al., 2013). Adiponectin plays a crucial role in down-regulating of hyperglycaemia and suppressing pancreatic islet apoptosis (Chai et al., 2011). A recent study indicates that adiponectin prevents pancreatic islet ischemia-reperfusion injury via inhibiting TNF-α production (Eizirik et al., 2009). Therefore, TNF-α and adiponectin are regarded as important factors in terms of β-cell apoptosis.

Sea cucumber is a popular marine food in China, and contains many kinds of bioactive compounds, such as sulphated polysaccharides, saponins, and lipids (Hu et al., 2013; Wu et al., 2013; Zhao et al., 2012; Zhong et al., 2007). Sulphated polysaccharides from sea cucumbers (namely fucoidan and fucosylated chondroitin sulphate) have attracted considerable attention in recent years. Current researches on bioactivities of fucoidan are mainly focused on anti-coagulation and anti-thrombosis (Chen et al., 2012), promotion of neural stem/progenitor cell proliferation (Zhang et al., 2010), alleviation of ethanol-induced gastric damage (Wang et al., 2012), inhibition of adipocyte differentiation (Kim et al., 2009) and osteoclastogenesis (Kariya et al., 2004). Our previous studies have shown that fucoidan could reduce blood glucose level and improves insulin resistance (Hu et al., 2014). However, its effect on pancreatic islets has not been understood. In the present study, we aimed at evaluating the effects of fucoidan isolated form Cucumaria frondosa (Cf-FUC) on inhibition of pancreatic islets apoptosis in insulin resistant mice and finding the mechanisms involved in the apoptosis-related genes expression in islets.

Materials and Methods

Materials    Dried Cucumaria frondosa was purchased at a seafood market in Qingdao, China. It was identified by Professor Yulin Liao of the Chinese Academy of Sciences Institute of Oceanography (Qingdao, China). Insulin and TNF-α ELISA assays kits, TRIzol reagent were Invitrogen products (Carlsbad, CA, USA). RNase free water, dNTPs, moloney murine leukemia virus reverse transcriptase (tumour), random primer, and PageRuler prestained protein ladder were from TaKaRa Bio Inc (Otsu, Shiga, Japan). Rabbit anti-rat t-Bid, Bax, cytochrome c, Bcl-2, Bcl-xL, caspase 3, cleaved-caspase 3, caspase 9, β-actin polyclonal antibodies, and goat-anti rabbit antibody IgG-HRP were Cell Signaling products (Beverly, MA, USA). Glucose test kit was from Biosino Bio-Tec (Beijing, China). Western blot IP lysis buffer, BCA protein concentration kit, and super-enhanced chemiluminescence (ECL) detection kit were Applygen Technologies Inc products (Beijing, China). The primers of Bid, Bax, Bak, Bcl-2, Bcl-xL, cytochrome c, caspase 9, caspase 3, and β-actin were synthesized by ShanGon Ltd. Co. (Shanghai, China).

Cf-FUC preparation    Cf-FUC was prepared from the body wall of wild C.frondosa as previously reported (Yu et al., 2013). The obtained Cf-FUC was composed of fucose, galactosamine, galactose and glucosamine with a ratio of 1:0.1:0.3:0.17. Its sulfat content was 29.31%. The protein content in Cf-FUC fraction was 7.84%.

Animals and experimental design    Four weeks old male C57BL/6J mice (18 – 20 g) were provided by Vital River Laboratory Animal Center (Beijing, China; Licensed ID: SCXK2009-0007). They were housed in a 12-h light/12-h dark cycle condition at 23 ± 1°C. The use of animals in this study was approved by the Ethical Committee of Experimental Animal Care at Ocean University of China. The insulin resistant model mice were established by fed a high-fat high-sucrose diet (HFSD, Research Diets, New Brunswick, NJ, USA; #D12331), which consisted 20% protein, 25% fat and 20% carbohydrates as previously described (HU et al., 2013). The mice were randomized into 6 groups of 10 animals each: control, HFSD-fed (HFSD), 1 mg/kg/d RSG-treated (RSG), 80 mg/kg/d Cf-FUC-treated (Cf-FUC), 20 mg/kg/d Cf-FUC plus 1 mg RSG/kg/d RSG-treated (20 Cf-FUC + RSG), and 80 mg/kg/d Cf-FUC plus 1 mg RSG/kg/d RSG-treated (80 Cf-FUC + RSG) groups. Mice in the control group were fed with a low-fat low-sucrose diet (LFSD, #D12328) and the others were fed with HFSD. After 19 weeks of treatment, the 8-h fasting animals were sacrificed. The blood was collected to test fasting blood glucose and adipokine levels. The tail of pancreas was cut off carefully to observe the histological structure of islets of langerhans and to detect apoptosis-related genes expression.

Blood glucose and insulin levels assay    Fasting blood glucose level was determined with a commercial kit (Biosino, Beijing, China). Serum insulin level was assessed with insulin ELISA assay kit (Invitrogen, Carlsbad, CA, USA). Insulin resistance was assessed by homeostasis model assessment of quantitative insulin sensitivity check index (QUICKI) and insulin resistance index (HOMA-IR) as follows(Bhuvaneswari and Anuradha, 2012):   

  

Serum adipokine determination    Serum adipokine levels, including adiponectin, resistin, leptin, and TNF-α, were determined by ELISA assay kits (Invitrogen, Carlsbad, CA, USA) according to the instructions.

Microscopic structures of islet of Langerhans    Pancreas tails were fixed in 10% formalin, paraffin embedded, sectioned, stained with hematoxylin and eosin (HE). Microscopic structure of islet of langerhans was photographed using an optical microscope (BH-2, Olympus, Japan).

RT-PCR analysis    The expression levels of apoptosis-related genes, namely Bid, Bax, Bcl-2, Bcl-xL, cytochrome c, caspase 9, and caspase 3 were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). RNA extract and amplification were performed according the methods described in the literatures with modification (Chang et al., 2012; Sikdar S, 2013; Srzentić et al., 2015). Briefly, the total RNA from gastrocnemius or adipose tissue was extracted using TRIzol reagent, and 1 µg RNA was reversely transcribed to cDNA with M-MLV. PCR was carried out in 30 µL system containing 0.5 µg cDNA on a MJ Research thermocycler (TC-96/G/H(b)A; Hangzhou, China). The thermocycling was performed by denaturing at 94°C for 45 s, annealing at the temperature desirable to each primer pair for 45 s and extending at 72°C for 45 s for cycling number set for each primer pair followed by an extra extending at 72°C for 10 min. PCR products were separated in 1% agarose gel buffered with Tris-acetate-EDTA and visualized with ethidium bromide staining. The housekeeping gene β-actin was used as a control. The parameters for RT-PCR are listed in Supplementary Table 1. Bands were quantitated using the Image J program (Version 1.41o; NIH, Bethesda, MA, USA). The mRNA relative expression levels were expressed as the ratio of signal intensity for the target genes to that of β-actin.

Supplementary Table 1. PCR primers used in apoptosis-related genes mRNA expression research

Genes	Primer	Tm (°C)	No. of cycle
Bid	F 5′-CCAAATAGGCGATGAGAT-3′ R 3′-CACCTGGAAATAGGGAGA-5′	55	30
Bax	F 5′-CTTCTTCCAGATGGTGAGTGAG-3′ R 3′-CCTTTTGCTTCAGGGTTTCA-5′	59	32
Cytochrome c	F 5′-GTCCTCGTCCGTGGTCGTT-3′ R 3′-TCAAGGTGGGCTGGCAAA-5′	55	34
Bcl-2	F 5′-TGGGAGAACAGGGTACGATA-3′ R 3′-CCACCGAACTCAAAGAAGG-5′	59	32
Bcl-xl	F 5′-ACCCCCTAAGAGCCATTT-3′ R 3′-GTCCCCTGAACCCACCTA-5′	58	32
Caspase 9	F 5′-ACAATAAATCTTCGGCAATAGG-3′ R 3′-TGGGGACCAGGCTCACTT-5′	52	34
Caspase 3	F 5′-TGACTGGAAAGCCGAAACT-3′ R 3′-CTGGATGAACCACGACCC-5′	52	36
β-actin	F 5′-GTTGGCGTTACACCCTTTCTTG-3′ R 3′-TGTCACCTTCACCGTTCCA-5′	60	27

Isolation of cytosolic fraction of pancreas    Cytosolic fraction of pancreas, which was used to measure cytochrome c release, was prepared following the reported method with modification (Karatug and Bolkent, 2013). Briefly, the pancreas tissues were disrupted by Western and IP lysis buffer including 50 mM Tris (pH 7.4), 150 mM NaCl, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM EDTA, 1 mM Na₃VO₄, and 0.5 µg/mL leupeptin in a glass homogenizer. The homogenate was centrifuged twice at 14,000×g for 30 min at 4°C. Then the supernatant was centrifuged at 100,000×g for 30 min at 4°C again. The yield cytosolic supernatant was used for assaying cytochrome c protein expression by western blot (WB).

WB analysis    To dissolve cellular protein, 0.1 g pancreas tails were lysed in Western and IP lysis buffer. The proteins were resolved by 10% SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes. Members blotted with 5% skimmed milk. Protein was incubated with t-Bid, Bax, cytochrome c, Bcl-2, Bcl-xL, caspase 3, cleaved-caspase 3, caspase 9, or β-actin primary antibodies and HRP-IGG secondary antibodies, and then was detected by ECL (Applygen, Beijing, China). Normalization of protein expression was assayed using β-actin as control.

Statistical analysis    Data were expressed as mean ± standard deviation (SD). Statistically comparisons were carried out by one-way analysis of variance (ANOVA) followed by Tukey's test using SPSS version 17.0 software. P<0.05 indicated statistically significant.

Results

Cf-FUC reduced blood glucose level and improved insulin resistance    Table 1 showed the blood glucose and serum insulin levels of the C57BL/6J mice. HFSD significantly increased fasting blood glucose, serum insulin, HOMA-IR, serum resistin and reduced QUICKI when compared to the control group (P < 0.01). Cf-FUC treatment decreased HFSD induced elevation in fasting blood glucose, serum insulin, HOMA-IR and serum resistin by 17.5%, 5.2%, 30.8% and 23.0%, and increased QUICKI by 4.5%, respectively. High dose Cf-FUC supplement enhanced the role of RSG in inhibiting the changes of fasting blood glucose, serum insulin, HOMA-IR, serum resistin and QUICKI caused by HFSD. These results suggest that Cf-FUC can significantly decrease blood glucose level and improve insulin resistance.

Table 1. Effects of Cf-FUC on the fasting blood glucose, insulin parameters and adipokines in HFSD-induced insulin resistant mice. ^a

Parameters	Control	HFSD	RSG	Cf-FUC	20Cf- FUC +RSG	80Cf- FUC +RSG
Fasting blood glucose (mmol/L)	9.27±1.40	13.12±0.82##	10.92±0.69*	10.82±1.41*	10.11±0.92*	9.72±1.24**, ⋆
Serum insulin (mU/L)	10.82±0.32	14.68±0.60##	13.36±0.14*	13.92±0.03*	13.01±0.19*	11.94±0.60**, ⋆⋆
HOMA-IR	4.53±0.31	8.22±0.95##	5.79±0.62**	5.69±0.57**	5.42±0.70**	4.56±0.48**, ⋆⋆
QUICKI	0.50±0.01	0.44±0.01##	0.47±0.00*	0.46±0.00*	0.47±0.00*	0.49±0.01**, ⋆
Serum resistin (ng/mL)	5.57±0.10	11.60±0.60##	8.43±0.18**	8.93±0.47**	9.40±0.28*	6.82±0.35**, ⋆⋆
Serum leptin (ng/mL)	0.14±0.01	0.32±0.03##	0.22±0.04*	0.19±0.00**	0.19±0.02**	0.14±0.01**, ⋆
Serum TNF-α (pg/mL)	57.0±1.4	100.8±6.5##	77.4±3.1**	80.3±4.5*	75.9±1.8**	66.6±1.4**, ⋆
Serum adiponectin (ng/mL)	205±10	110±8##	152±7**	137±4*	167±5**	186±8**, ⋆⋆

^a  Data are presented as mean±SD (n=10/group). Multiple comparisons were done using one way ANOVA analysis followed by Tukey's test.

^##  P < 0.01 versus the control group.

*  P < 0.05,

**  P < 0.01 versus the HFSD mice.

⋆  P < 0.05,

⋆⋆  P < 0.01 versus the RSG-treated mice.

Cf-FUC decreased TNF-α level and increased adiponectin level    The inflammatory mediator, TNF-α, is involved in apoptosis of pancreatic acinar cells (Liu et al., 2012). Adiponectin is reported as an adipocytokine that can reduce TNF-α production and inhibit pancreatic islets apoptosis (Du et al., 2013). Table 1 showed that Cf-FUC significantly suppressed both HFSD-induced TNF-α increase and adiponectin reduction. High-dose Cf-FUC further decreased serum TNF-α level and increased adiponectin level when combined with RSG. These results suggest that Cf-FUC can significantly reduce the inflammatory factor in insulin resistant mice.

Cf-FUC repaired islet of Langerhans    Fig. 1 showed that various pathologic manifestations such as cell necrosis, atrophy of islet of langerhans, substantial inflammatory infiltration, and nuclear chromatin uneven distribution were observed in islet of langerhans of HFSD mice. These histological changes were alleviated in Cf-FUC-treated mice. When the mice were treated by Cf-FUC + RSG combination, the pathological alleviation was better than that in RSG group. High dose Cf-FUC combined with RSG further normalized the histology, which was approximated to that in the control group. These results suggest that Cf-FUC can repair HFSD-injured pancreatic islets.

Fig. 1.

Effect of Cf-FUC on microstructure of islets of langerhans in insulin resistant mice. (× 400; arrow points to islets of langerhans, n=4/group)

Cf-FUC decreased caspase 3 and caspase 9 expression    To clarify whether Cf-FUC repaired HFSD-injured pancreatic islets through inhibiting islets cells apoptosis, caspase 3 and caspase 9 genes expression levels were studied. Caspases play essential roles in apoptosis. Caspase 3 is regarded as an apoptosis marker, and caspase 9 is an initiator of caspase 3 activity (Hou et al., 2013; Saleh et al., 2014). As shown in Fig. 2, Cf-FUC significantly reduced the mRNA expression level of caspase 9 in HFSD-fed mice pancreatic islets (P < 0.05), but caspase 3 mRNA expression unchanged. Although the protein expressions of caspase 9 and cleaved-caspase 3 were decreased in Cf-FUC group, there was no statistically significant. RSG-treated group showed significant decrease in the mRNA expression levels of caspase 9 and caspase 3, and in the protein expression levels of caspase 9 and cleaved caspase 3. These suggest that the activity of RSG was better than Cf-FUC. It is dramatical that the mRNA expression levels of caspase 9 and caspase 3 in 80 Cf-FUC+RSG group were lower than those in RSG group (P < 0.05). Moreover, the same changes were shown in the protein expression levels (P < 0.01). These results suggest that Cf-FUC can synergistically enhance the effect of RSG on inhibiting pancreatic islets apoptosis.

Fig. 2.

Effects of Cf-FUC on the mRNA and protein expression of caspase 9 and caspase 3 in pancreatic islets of insulin resistant mice. The same photos of β-actin in mRNA and protein expression were used. A: caspase 9 mRNA expression, B: caspase 9 protein expression, C: caspase 3 mRNA expression, D: cleaved-caspase 3 protein expression. Data are expressed as mean ± S.D. (n=6/group). Multiple comparisons were done using one way ANOVA analysis followed by Tukey's test. ^##P < 0.01 versus control; *P < 0.05, **P < 0.01 versus HFSD; ⋆P < 0.05, ⋆⋆P < 0.01 versus RSG group.

Cf-FUC inhibited intrinsic mitochondrial pathway    As Cf-FUC significantly suppressed apoptosis in pancreatic islets, we examined its effects on mitochondrial pathway which was the pivotal signaling of cell apoptosis (Xiang et al., 2015). Cytochrome c release is recognized as a key point in this mitochondrial pathway (Zhu et al., 2015). As shown in Fig. 3, HFSD-induced cytochrome c mRNA expression elevation in the pancreatic islets was reduced by Cf-FUC to 74.8% (P < 0.05). Further, Cf-FUC significantly decreased cytochrome c protein level in cytoplasm by 19.6% (P < 0.05), which indicated that Cf-FUC inhibited mitochondrial cytochrome c release to the cytoplasm. These beneficial effects were further improved when high dose Cf-FUC combined with RSG (P < 0.05, P < 0.01). Bax plays an essential role in the activation of the execution phase of apoptosis by binding to and antagonizing the Bcl-2 protein (Feng et al., 2015; Mignard et al., 2014). Bid, a BH3 only protein, is cleaved to t-bid by activated caspase 8 and subsequently induces Bax assemblage from cytoplasm to mitochondria (Hutcheson, & Perlman, 2008). As shown in Fig. 4, Cf-FUC significantly lowered both Bax and Bid mRNA expression levels in the pancreatic islets of insulin resistant mice (P < 0.05). WB results further indicated that Bax and t-Bid protein expression levels were significantly reduced by 26.6 and 34.9% when treated with Cf-FUC (P < 0.05, P < 0.01), respectively. Both Bcl-2 and Bcl-xL can reduce caspase activity and inhibit cell apoptotic processes (Shi et al., 2015). As shown in Fig. 5, mRNA expression levels of Bcl-2 and Bcl-xL were 1.49-fold and 1.74-fold in Cf-FUC-treated mice than those in HFSD-fed animals, respectively, though there was no statistically significant in Bcl-xL mRNA expression. WB assays further showed that Cf-FUC significantly increased these two protein expression levels by 1.60-fold and 1.74-fold compared to that of HFSD-fed mice (P < 0.05, P < 0.01). Moreover, the mRNA expression of Bcl-xL and the protein expression of Bcl-2 and Bcl-xL were all significant increased in 80 Cf-FUC+RSG group compared with RSG alone group (P < 0.05, P < 0.01). These results suggest that Cf-FUC can suppress pancreatic islets apoptosis via blocking intrinsic mitochondrial pathway, though its effects were lower than RSG. Cf-FUC can also enhance the effects of RSG when they were combined.

Fig. 3.

Effects of Cf-FUC on mRNA and protein expression of cytochrome c in pancreatic islets of insulin resistant mice. Cytochrome c protein was detected using the cytosol fraction of pancreatic islets. The same photos of β-actin in mRNA and protein expression were used. A: cytochrome c mRNA expression, B: cytochrome c protein expression. Data are expressed as mean ± S.D. (n=6/group). Multiple comparisons were done using one way ANOVA analysis followed by Tukey's test. ^##P < 0.01 versus control; *P < 0.05, **P < 0.01 versus HFSD; ⋆P < 0.05 versus RSG group.

Fig. 4.

Effects of Cf-FUC on mRNA and protein expression of Bax and Bid in pancreatic islets of insulin resistant mice. The same photos of β-actin in mRNA and protein expression were used. A: Bax mRNA expression, B: Bax protein expression, C: Bid mRNA expression, D: t-Bid protein expression. Data are expressed as mean ± S.D. (n=6/group). Multiple comparisons were done using one way ANOVA analysis followed by Tukey's test. ##P < 0.01 versus control; *P < 0.05, **P < 0.01 versus HFSD; ⋆P < 0.05 versus RSG group.

Fig. 5.

Effects of Cf-FUC on mRNA and protein expression of Bcl-2 and Bcl-xL in pancreatic islets of insulin resistant mice. The same photos of β-actin in mRNA and protein expression were used. A: Bcl-2 mRNA expression, B: Bcl-2 protein expression, C: Bcl-xL mRNA expression, D: Bcl-xL protein expression. Data are expressed as mean ± S.D. (n=6/group). Multiple comparisons were done using one way ANOVA analysis followed by Tukey's test. ^##P < 0.01 versus control; *P < 0.05, **P < 0.01 versus HFSD; ⋆P < 0.05, ⋆⋆P < 0.01 versus RSG group.

Discussion

The results of this study demonstrated that Cf-FUC could improve hyperglycemia and increase insulin sensitivity in HFSD-induced insulin resistant mice. Our data further suggested that Cf-FUC could suppress pancreatic islets apoptosis by reducing TNF-α level and increasing adiponectin level. Here, we reported for the first time that the inhibition of apoptosis in islets of langerhans by Cf-FUC depended on the down regulation of mitochondrial pathway.

It has been reported that inflammatory cytokines can induce the death of pancreatic β-cell (Govindaraj and Sorimuthu Pillai, 2015; Tian et al., 2014). The present study indicated that serum level of the pro-inflammatory cytokines, TNF-α, was significantly higher in the HFSD group than that in the control group. Cf-FUC supplement could significantly decrease serum TNF-α level of the HFSD-treated mice. Adiponectin is an adipose tissue-derived mediator with anti-inflammatory and insulin sensitizing properties (Ziemke and Mantzoros, 2010). Previous studies have revealed that adiponectin can suppress glucotoxicity-induced cell apoptosis (Lindmark et al., 2006; Lin et al., 2009). Du et al. (2013) and Thole et al. (2012) reported that TNF-α exacerbated impaired insulin action and destruction of pancreatic β-cell. In this study, histological changes showed that Cf-FUC repaired the injury of the islet of langerhans, indicating that Cf-FUC could protect pancreas cells from death by reducing TNF-α level in insulin resistant mice.

Cieślak et al. (2015) reported that biological activities of TNF-α were exerted through mitochondrial dysfunction and activation of various downstream signaling effectors. These effectors led to the activation of specific signaling pathways of apoptosis, such as the release of mitochondrial cytochrome c into the cytoplasm (Yang et al., 2014). In this study, HFSD induced a significant increase of cytochrome c mRNA expression in the pancreatic islets, but Cf-FUC could alleviate this induction. Moreover, Cf-FUC also significantly decreased the release of cytochrome c to cytoplasm. Once entering the cytoplasm, cytochrome c could directly activate caspases, which cleaved a series of cellular proteins and thus caused apoptotic changes. Some studies demonstrated that the reduction of cytochrome c release could cause the inactivation of apoptosis related effectors, such as caspase 3 and caspase 9 (Bishayee et al., 2013; Jiang et al., 2013). Our research results were consistent with the previous findings, which showed that caspase 3 and caspase 9 mRNA expression levels were down-regulated, and caspase 3 protein and cleaved-caspase 9 protein expression levels were also reduced in HFSD-treated mice by Cf-FUC. Therefore, it could be speculated that Cf-FUC supplement inhibited cytochrome c release, and subsequently suppressed the activation of caspase 3 and caspase 9, which finally blocked the mitochondrial apoptotic pathway.

Cytochrome c release and mitochondria disruption are controlled by the Bcl-2 family proteins (Vander Heiden and Thompson, 1999). Bcl-2 and Bcl-xL inhibit apoptosis by cleaning up reactive oxygen species, preventing the release of Ca²⁺ from the endoplasmic reticulum and preventing the mitochondrial transitional pore opening by opposing the effect of Bax thereby blocking cytochrome c release and inhibiting caspase activities (Bouzakri et al., 2011; Donovan and Cotter, 2004; Lamothe and Aggarwal, 2002; Sha et al., 2008). In our present study, Bcl-2 and Bcl-xL were significantly decreased by HFSD treatment, but these decreases could be inhibited by Cf-FUC. The results indicated that Bcl-2 family proteins might play a critical role in regulating mitochondrial pathway of cytochrome c release, and Cf-FUC was able to protect pancreatic islets from apoptosis via modulating of Bcl-2 and Bcl-xL expression levels. The key pro-apoptotic protein, Bax, which is a main inhibitor of Bcl-2 and Bcl-xL, is one member of the apoptosis-regulated gene Bcl-2 family (Antonsson et al., 1997). Upon activation, Bax can induce cytochrome c release to cytoplasm by multiple mechanisms (Eskes et al., 1998; Gómez-Crisóstomo et al., 2013). In this study, Cf-FUC significantly inhibited both mRNA and protein expression levels of Bax in HFSD-treated mice. Caspase 8 mediates the intrinsic pathway through cleavage of the pro-apoptotic Bid protein, a BH3-only protein, to a truncated Bid (t-Bid) via translocation from the cytoplasm to the mitochondria, triggering mitochondrial dysfunction (Hutcheson and Perlman, 2009; Park et al., 2013). This always leads to the activation of Bax and opening of mitochondrial membrane (Shamas-Din et al., 2013; Strasser, 2005). It was shown in our research that Cf-FUC significantly decreased Bid mRNA and t-Bid protein expression levels. These results demonstrated that Cf-FUC suppressed pancreatic islets apoptosis via inactivation of Bid protein and consequent inactivation of the intrinsic mitochondrial pathway. The reduction of Bax, activation of Bcl-2 and Bcl-xL by Cf-FUC further inhibited cytochrome c release to the cytoplasm and subsequently inactivated the apoptotic effectors caspase 9 and caspase 3.

FUC from some marine plants was also proved to exhibit the activities of improving insulin resistance and providing pancreatic protection. Jiang et al. (2015) reported that FUC from the sea weed Fucus vesiculosus could stimulate insulin secretion and provide pancreatic protection via the cyclic adenosine monophosphate (cAMP) pathway in RIN-5F rat insuin-secreting cells and GK rats. Hernández-Corona et al. (2014) reported that FUC from marine plants could promote insulin secretion and improve insulin resistance in overweight or obese adults. Carvalho et al. (2014) proved that FUC reduced the severity of acute pancreatitis in mice by decreasing neutrophil infiltration and systemic inflammation. Our results showed that Cf-FUC also increased serum insulin level, reduced inflammatory cytokines levels, and protected pancreatic islets apoptosis. It was implied to be concerned with the activities of inhibition of α-amylase and α-glucosidase by FUC derived from brown marine algae (Kim, Rioux and Turgeon, 2014). They also reported that the activities of FUC were different from the distinct species.

In the present study, the dosage of Cf-FUC was also examined. The protection effects of structure and insulin secretion of islets in (80 Cf-FUC + RSG)-treated group were significantly improved when compared with the RSG-treated group. When compared with the RSG-treated group, the mitochondrial pathway was significantly inactivated in (80 Cf-FUC + RSG)-treated group but not in (20 Cf-FUC +RSG)-treated group. These results indicate that the dosage of Cf-FUC was crucial for its effect on protecting islets of langedrhans from apoptosis, and it was effective when the dosage of Cf-FUC was up to 80 mg/kg/d. On the other hand, these study were not investigate the effects of a dose dependency of RSG and fucoidan combination on insulin resistance and pancreas apoptosis, and the future study will be focused on it.

Conclusion

In the present study, the results demonstrated that Cf-FUC could inhibit HFSD-induced pancreatic islets apoptosis. Activation of Bcl-2 and Bcl-xL, which led to the inactivation of intrinsic mitochondrial pathway, was the underlying mechanism for the protection effects of Cf-FUC. Thus, supplementation with Cf-FUC gives a dietary intervention for pancreatic islets apoptosis development, providing an alternative precaution for insulin resistance or type 2 diabetes.

Acknowledgements    This study was supported by National Natural Science Foundation of China (31071525) and Research Start-up Funding of Zhejiang Ocean University (Q1442 and Q1443). We also thank Yulin Liao (the Chinese Academy of Sciences Institute of Oceanography, Qingdao, China) for his species identification of the sea cucumber Cusumaria frondosa.

Abbreviations

ANOVA

one-way analysis of variance

Cf-FUC

Fucoidan from Cucumaria frondosa

ECL

super-enhanced chemiluminescence

HE

hematoxylin and eosin

HFSD

high-fat high-sucrose diet

HOMA-IR

homeostasis model assessment of insulin resistance index

LFSD

low-fat low-sucrose diet

M-MLV

moloney murine leukemia virus

PVDF

polyvinylidene fluoride

QUICKI

quantitative insulin sensitivity check index

RSG

rosiglitazone

RT-PCR

reverse transcriptase-polymerase chain reaction

SD

standard deviation

References

•  Alberti,  K.G. and  Zimmet,  P.Z. (1998). Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: diagnosis and classification of diabetes mellitus. Provisional report of a WHO Consultation. Diabet. Med., 15, 539-553.
•  Antonsson,  B.,  Conti,  F.,  Ciavatta,  A.,  Montessuit,  S.,  Lewis,  S.,  Martinou,  I.,  Bernasconi,  L.,  Bernard,  A.,  Mermod,  J.J.,  Mazzei,  G.,  Maundrell,  K.,  Gambale,  F.,  Sadoul,  R., and  Martinou,  J.C. (1997). Inhibition of Bax Channel-Forming Activity by Bcl-2. Science, 277, 370-372.
•  Bhuvaneswari,  S.. and  Anuradha,  C.V. (2012). Astaxanthin prevents loss of insulin signaling and improves glucose metabolism in liver of insulin resistant mice. Can. J. Physiol. Pharmacol, 90, 1544-1552.
•  Bishayee,  K.,  Ghosh,  S.,  Mukherjee,  A.,  Sadhukhan,  R.,  Mondal,  J., and  Khuda-Bukhsh,  A.R. (2013). Quercetin induces cytochrome-c release and ROS accumulation to promote apoptosis and arrest the cell cycle in G2/M, in cervical carcinoma: signal cascade and drug-DNA interaction. Cell Prolif., 46, 153-163.
•  Blesso,  C.N.,  Andersen,  C.J.,  Barona,  J.,  Volk,  B.,  Volek,  J.S., and  Fernandez,  M.L. (2013). Effects of carbohydrate restriction and dietary cholesterol provided by eggs on clinical risk factors in metabolic syndrome. J. Clin. Lipidol., 7, 463-471.
•  Bouzakri,  K.,  Plomgaard,  P.,  Berney,  T.,  Donath,  M.Y.,  Pedersen,  B.K., and  Halban,  P.A. (2011). Bimodal effect on pancreatic β-cells of secretory products from normal or insulin-resistant human skeletal muscle. Diabetes, 60, 1111-1121.
•  Cai,  K.,  Qi,  D.,  Wang,  O.,  Chen,  J.,  Liu,  X.,  Deng,  B.,  Qian,  L.,  Liu,  X., and  Le,  Y. (2011). TNF-α acutely upregulates amylin expression in murine pancreatic beta cells. Diabetologia, 54, 617-626.
•  Carvalho,  A.C.,  Sousa,  R.B.,  Franco,  Á.X.,  Costa,  J.V.,  Neves,  L.M.,  Ribeiro,  R.A.,  Sutton,  R.,  Criddle,  D.N.,  Soares,  P.M., and  de Souza,  M.H. (2014). Protective effects of fucoidan, a P- and L-selectin inhibitor, in murine acute pancreatitis. Pancreas, 43, 82-87.
•  Chai,  F.,  Wang,  Y.,  Zhou,  Y.,  Liu,  Y.,  Geng,  D., and  Liu,  J. (2011). Adiponectin downregulates hyperglycemia and reduces pancreatic islet apoptosis after roux-en-y gastric bypass surgery. Obes. Surg., 21, 768-773.
•  Chang,  C.Y.,  Lee,  T.-H., and  Sheu,  W.H.H. (2012). Anti-atherogenic effects of resveratrol via liver X receptor α-dependent upregulation of ATP-binding cassette transporters A1 and G1 in macrophages. J. Funct. Foods, 4, 727-735.
•  Chen,  X.,  Yang,  L., and  Zhai,  S. (2012). Risk of cardiovascular disease and all-cause mortality among diabetic patients prescribed rosiglitazone or pioglitazone: a meta-analysis of retrospective cohort studies. Chin. Med. J. (Engl.), 125, 4301-4306.
•  Cieślak,  M.,  Wojtczak,  A., and  Cieślak,  M. (2015). Role of pro-inflammatory cytokines of pancreatic islets and prospects of elaboration of new methods for the diabetes treatment. Acta Biochim. Pol., 62, 15-21.
•  Donovan,  M. and  Cotter,  T.G. (2004). Control of mitochondrial integrity by Bcl-2 family members and caspase-independent cell death. Biochim. Biophys. Acta BBA - Mol. Cell Res., 1644, 133-147.
•  Du,  X.,  He,  S.,  Jiang,  Y.,  Wei,  L., and  Hu,  W. (2013). Adiponectin prevents islet ischemia-reperfusion injury through the COX2-TNFα-NF-κB-dependent signal transduction pathway in mice. J. Endocrinol., 218, 75- 84.
•  Eizirik,  D.L.,  Colli,  M.L., and  Ortis,  F. (2009). The role of inflammation in insulitis and beta-cell loss in type 1 diabetes. Nat. Rev. Endocrinol., 5, 219-226.
•  Eskes,  R.,  Antonsson,  B.,  Osen-Sand,  A.,  Montessuit,  S.,  Richter,  C.,  Sadoul,  R.,  Mazzei,  G.,  Nichols,  A., and  Martinou,  J.C. (1998). Bax-induced cytochrome C release from mitochondria is independent of the permeability transition pore but highly dependent on Mg²⁺ ions. J. Cell Biol., 143, 217-224.
•  Feng,  Y.,  Xu,  X.,  Zhang,  Y.,  Ding,  J.,  Wang,  Y.,  Zhang,  X.,  Wu,  Z.,  Kang,  L.,  Liang,  Y.,  Zhou,  L.,  Song,  S.,  Zhao,  K., and  Ye,  Q. (2015). HPIP is upregulated in colorectal cancer and regulates colorectal cancer cell proliferation, apoptosis and invasion. Sci. Rep., 5, 9429.
•  Gómez-Crisóstomo,  N.P.,  López-Marure,  R.,  Zapata,  E.,  Zazueta,  C., and  Martínez-Abundis,  E. (2013). Bax induces cytochrome c release by multiple mechanisms in mitochondria from MCF7 cells. J. Bioenerg. Biomembr., 45, 441-448.
•  Govindaraj,  J. and  Sorimuthu Pillai,  S. (2015). Rosmarinic acid modulates the antioxidant status and protects pancreatic tissues from glucolipotoxicity mediated oxidative stress in high-fat diet: streptozotocin-induced diabetic rats. Mol. Cell. Biochem., in press
•  Hernández-Corona,  D.M.,  Martínez-Abundis,  E., and  González-Ortiz,  M. (2014). Effect of fucoidan administration on insulin secretion and insulin resistance in overweight or obese adults. J. Med. Food, 17, 830-832.
•  Hou,  G.,  Li,  C.,  Liu,  S.,  Huan,  Y.,  Liu,  Q.,  Sun,  S.,  Li,  L.,  Hou,  S., and  Shen,  Z. (2013). Long-term treatment with EXf, a peptide analog of Exendin-4, improves β-cell function and survival in diabetic KKAy mice. Peptides, 40, 123-132.
•  Hu,  S.,  Chang,  Y.,  Wang,  J.,  Xue,  C.,  Li,  Z., and  Wang,  Y. (2013). Fucosylated Chondroitin Sulfate from Sea Cucumber in Combination with Rosiglitazone Improved Glucose Metabolism in the Liver of the Insulin-Resistant Mice. Biosci. Biotechnol. Biochem., 77, 2263-2268.
•  Hu,  S.,  Chang,  Y.,  Wang,  J.,  Xue,  C.,  Shi,  D.,  Xu,  H., and  Wang,  Y. (2013). Fucosylated chondroitin sulfate from Acaudina molpadioides improves hyperglycemia via activation of PKB/GLUT4 signaling in skeletal muscle of insulin resistant mice. Food Funct., 4, 1639-1646.
•  Hu,  S.,  Xia,  G.,  Wang,  J.,  Wang,  Y.,  Li,  Z., and  Xue,  C. (2014). Fucoidan from sea cucumber protects against high-fat high-sucrose diet-induced hyperglycaemia and insulin resistance in mice. J. Funct. Foods, 10, 128-138.
•  Hutcheson,  J. and  Perlman,  H. (2009). BH3-only proteins in rheumatoid arthritis: potential targets for therapeutic intervention. Oncogene, 27, S168-S175.
•  J E Shaw,  R.A.S. (2009). Global estimates of the prevalence of diabetes for 2010 and 2030. Diabetes Res. Clin. Pract., 87, 4-14.
•  Jiang,  X.,  Yu,  J.,  Ma,  Z.,  Zhang,  H., and  Xie  F. (2015). Effects of fucoidan on insulin stimulation and pancreatic protection via the cAMP signaling pathway in vivo and in vitro. Mol. Med. Rep., 12, 4501-4507.
•  Jiang,  Y.,  Yang,  J.,  Yang,  C.,  Meng,  F.,  Zhou,  Y.,  Yu,  B.,  Khan,  M., and  Yang,  H. (2013). Vitamin K4 induces tumor cytotoxicity in human prostate carcinoma PC-3 cells via the mitochondria-related apoptotic pathway. Pharm., 68, 442-448.
•  Jurgens,  C.A.,  Toukatly,  M.N.,  Fligner,  C.L.,  Udayasankar,  J.,  Subramanian,  S.L.,  Zraika,  S.,  Aston-Mourney,  K.,  Carr,  D.B.,  Westermark,  P.,  Westermark,  G.T.,  Kahn,  S.E., and  Hull,  R.L. (2011). β-Cell Loss and β-Cell Apoptosis in Human Type 2 Diabetes Are Related to Islet Amyloid Deposition. Am. J. Pathol., 178, 2632-2640.
•  Kang,  J.-H.,  Tsuyoshi,  G.,  Han,  I.-S.,  Kawada,  T.,  Kim,  Y.M., and  Yu,  R. (2010). Dietary Capsaicin Reduces Obesity-induced Insulin Resistance and Hepatic Steatosis in Obese Mice Fed a High-fat Diet. Obesity, 18, 780-787.
•  Karatug,  A. and  Bolkent,  S. (2013). The potential role of combined antioxidant treatment on pancreas of STZ-diabetic mice. Exp. Toxicol. Pathol., 65, 255-262.
•  Kariya,  Y.,  Mulloy,  B.,  Imai,  K.,  Tominaga,  A.,  Kaneko,  T.,  Asari,  A.,  Suzuki,  K.,  Masuda,  H.,  Kyogashima,  M., and  Ishii,  T. (2004). Isolation and partial characterization of fucan sulfates from the body wall of sea cucumber Stichopus japonicus and their ability to inhibit osteoclastogenesis. Carbohydr. Res., 339, 1339-1346.
•  Kim,  K.T.,  Rioux,  L.E., and  Turgeon,  S.L. (2014). Alpha-amylase and alpha-glucosidase inhibition is differentially modulated by fucoidan obtained from Fucus vesiculosus and Ascophyllum nodosum. Phytochemistry, 98, 27-33.
•  Kim,  M.J.,  Chang,  U.J., and  Lee,  J.S. (2009). Inhibitory effects of Fucoidan in 3T3-L1 adipocyte differentiation. Mar. Biotechnol. N. Y. N, 11, 557-562.
•  Lamothe,  B. and  Aggarwal,  B.B. (2002). Ectopic expression of Bcl-2 and Bcl-xL inhibits apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) through suppression of caspases-8, 7, and 3 and BID cleavage in human acute myelogenous leukemia cell line HL-60. J. Interferon Cytokine Res. 22, 269-279.
•  Lee,  E.,  Ryu,  G.R.,  Ko,  S.H.,  Ahn,  Y.B.,  Yoon,  K.H.,  Ha,  H., and  Song,  K.H. (2011). Antioxidant treatment may protect pancreatic beta cells through the attenuation of islet fibrosis in an animal model of type 2 diabetes. Biochem. Biophys. Res. Commun., 414, 397-402.
•  Lee,  J. (2013). Adipose tissue macrophages in the development of obesity-induced inflammation, insulin resistance and type 2 Diabetes. Arch. Pharm. Res., 36, 208-222.
•  Lindmark,  S.,  Burén,  J., and  Eriksson,  J.W. (2006). Insulin resistance, endocrine function and adipokines in type 2 diabetes patients at different glycaemic levels: potential impact for glucotoxicity in vivo. Clin. Endocrinol. (Oxf.), 65, 301-309.
•  Lin,  P.,  Chen,  L.,  Li,  D.,  Liu,  J.,  Yang,  N.,  Sun,  Y.,  Xu,  Y.,  Fu,  Y., and  Hou,  X. (2009). Adiponectin reduces glucotoxicity-induced apoptosis of INS-1 rat insulin-secreting cells on a microfluidic chip. Tohoku J. Exp. Med., 217, 59-65.
•  Liu,  Y.,  Zhou,  Z.G.,  Chen,  K.L.,  Zhou,  B.,  Yang,  L.,  Yan,  H., and  Li,  Y. (2012). The ER chaperone GRP78 is associated with the severity of cerulein-induced pancreatic inflammation via regulating apoptosis of pancreatic acinar cells. Hepatogastroenterology, 59, 1670-1676.
•  Maedler,  K. (2008). Beta cells in type 2 diabetes - a crucial contribution to pathogenesis. Diabetes Obes. Amp Metab., 10, 408-20.
•  Marselli,  L.,  Bugliani,  M.,  Suleiman,  M.,  Olimpico,  F.,  Masini,  M.,  Petrini,  M.,  Boggi,  U.,  Filipponi,  F.,  Syed,  F., and  Marchetti,  P. (2013). β-Cell inflammation in human type 2 diabetes and the role of autophagy. Diabetes Obes. Metab., 15 Suppl 3, 130-136.
•  Meier,  J.J. and  Bonadonna,  R.C. (2013). Role of Reduced β-Cell Mass Versus Impaired β-Cell Function in the Pathogenesis of Type 2 Diabetes. Diabetes Care, 36, S113-S119.
•  Mignard,  V.,  Lalier,  L.,  Paris,  F., and  Vallette,  F.M. (2014). Bioactive lipids and the control of Bax pro-apoptotic activity. Cell Death Dis., 5, e1266.
•  Moon,  H.,  Chon,  J.,  Joo,  J.,  Kim,  D.,  In,  J.,  Lee,  H.,  Park,  J., and  Choi,  J. (2013). FTY720 preserved islet β -cell mass by inhibiting apoptosis and increasing survival of β -cells in db/db mice: FTY720 Preserves Islet β -cell in Mice. Diabetes Metab. Res. Rev., 29, 19-24.
•  Muoio,  D.M. and  Newgard,  C.B. (2008). Mechanisms of disease:Molecular and metabolic mechanisms of insulin resistance and beta-cell failure in type 2 diabetes. Nat. Rev. Mol. Cell Biol., 9, 193-205.
•  Park,  H.S.,  Hwang,  H.J.,  Kim,  G.Y.,  Cha,  H.-J.,  Kim,  W.J.,  Kim,  N.D.,  Yoo,  Y.H., and  Choi,  Y.H. (2013). Induction of apoptosis by fucoidan in human leukemia U937 cells through activation of p38 MAPK and modulation of Bcl-2 family. Mar. Drugs, 11, 2347-2364.
•  Saleh,  A.M.,  Aljada,  A.,  Rizvi,  S.A.,  Nasr,  A.,  Alaskar,  A.S., and  Williams,  J.D. (2014). In vitro cytotoxicity of Artemisia vulgaris L. essential oil is mediated by a mitochondria-dependent apoptosis in HL-60 leukemic cell line. BMC Complement. Altern. Med., 14, 226.
•  Sha,  H.,  Ma,  Q.,  Jha,  R.K.,  Xu,  F.,  Wang,  L.,  Wang,  Z.,  Zhao,  Y., and  Fan,  F. (2008). Resveratrol ameliorates hepatic injury via the mitochondrial pathway in rats with severe acute pancreatitis. Eur. J. Pharmacol., 601, 136-142.
•  Shamas-Din,  A.,  Bindner,  S.,  Zhu,  W.,  Zaltsman,  Y.,  Campbell,  C.,  Gross,  A.,  Leber,  B.,  Andrews,  D.W., and  Fradin,  C. (2013). tBid Undergoes Multiple Conformational Changes at the Membrane Required for Bax Activation. J. Biol. Chem., 288, 22111-22127.
•  Shi,  M.,  He,  X.,  Wei,  W.,  Wang,  J.,  Zhang,  T., and  Shen,  X. (2015). Tenascin-C induces resistance to apoptosis in pancreatic cancer cell through activation of ERK/NF-κB pathway. Apoptosis Int. J. Program. Cell Death., In press
•  Sikdar  S, M.A. (2013). Post-cancer treatment with Condurango 30C shows amelioration of banzo[a]pyrene-induced lung cancer of rats through Caspase-3 mediated apoptosis induction. J Pharmacopuncture, 16, 11-22.
•  Srzentić,  S.,  Nikčević,  G.,  Spasovski,  D.,  Baščarević,  Z.,  Živković,  Z.,  Terzic-Šupić,  Z.,  Matanović,  D.,  Djordjević,  V.,  Pavlović,  S., and  Spasovski,  V. (2015). Predictive genetic markers of coagulation, inflammation and apoptosis in Perthes disease—Serbian experience. Eur. J. Pediatr., 174, 1085-1092.
•  Strasser,  A. (2005). The role of BH3-only proteins in the immune system. Nat. Rev. Immunol., 5, 189-200.
•  Thole,  A.A.,  Rodrigues-Cunha,  A.C.S.,  Carvalho,  S.N.,  Garcia-Souza,  E.P.,  Cortez,  E.,  Stumbo,  A.C.,  Carvalho,  L., and  Moura,  A.S. (2012). Progenitor cells and TNF-alpha involvement during morphological changes in pancreatic islets of obese mice. Tissue Cell, 44, 238-248.
•  Tian,  Y.F.,  Chang,  W.C.,  Loh,  C.H., and  Hsieh,  P.S. (2014). Leptin-mediated inflammatory signaling crucially links visceral fat inflammation to obesity-associated β-cell dysfunction. Life Sci., 116, 51-58.
•  Vander Heiden,  M.G. and  Thompson,  C.B. (1999). Bcl-2 proteins: regulators of apoptosis or of mitochondrial homeostasis? Nat. Cell Biol., 1, E209-E216.
•  Wang,  Y.,  Su,  W.,  Zhang,  C.,  Xue,  C.,  Chang,  Y.,  Wu,  X.,  Tang,  Q., and  Wang,  J. (2012). Protective effect of sea cucumber (Acaudina molpadioides) fucoidan against ethanol-induced gastric damage. Food Chem., 133, 1414-1419.
•  Wu,  F.-J.,  Xue,  Y.,  Tang,  Q.-J.,  Xu,  J.,  Du,  L.,  Xue,  C.-H.,  Takahashi,  K., and  Wang,  Y.-M. (2013). Protective effects of cerebrosides from sea cucumber and starfish on the oxidative damage in PC12 cells. J. Oleo Sci., 62, 717-727.
•  Xiang,  R.L.,  Mei,  M.,  Su,  Y.C.,  Li,  L.,  Wang,  J.Y., and  Wu,  L.L. (2015). Visfatin Protects Rat Pancreatic β-cells against IFN-γ-Induced Apoptosis through AMPK and ERK1/2 Signaling Pathways. Biomed. Environ. Sci. BES, 28, 169-177.
•  Yang,  L.,  Rong,  Z.,  Zeng,  M.,  Cao,  Y.,  Gong,  X.,  Lin,  L.,  Chen,  Y.,  Cao,  W.,  Zhu,  L., and  Dong,  W. (2015). Pyrroloquinoline quinone protects nucleus pulposus cells from hydrogen peroxide-induced apoptosis by inhibiting the mitochondria-mediated pathway. Eur. Spine J. 24, 1702-1710.
•  Yu,  L.,  Xu,  X.,  Xue,  C.,  Chang,  Y.,  Ge,  L.,  Wang,  Y.,  Zhang,  C.,  Liu,  G., and  He,  C. (2013). Enzymatic preparation and structural determination of oligosaccharides derived from sea cucumber (Acaudina molpadioides) fucoidan. Food Chem., 139, 702-709.
•  Zhang,  Y.,  Song,  S.,  Song,  D.,  Liang,  H.,  Wang,  W., and  Ji,  A. (2010). Proliferative effects on neural stem/progenitor cells of a sulfated polysaccharide purified from the sea cucumber Stichopus japonicus. J. Biosci. Bioeng., 109, 67-72.
•  Zhang,  Z.,  Ding,  Y.,  Dai,  X.,  Wang,  J., and  Li,  Y. (2011). Epigallocatechin-3-gallate protects pro-inflammatory cytokine induced injuries in insulin-producing cells through the mitochondrial pathway. Eur. J. Pharmacol., 670, 311-316.
•  Zhao,  Q.,  Xue,  Y.,  Wang,  J.,  Li,  H.,  Long,  T.,  Li,  Z.,  Wang,  Y.,  Dong,  P., and  Xue,  C. (2012). In vitro and in vivo anti-tumour activities of echinoside A and ds-echinoside A from Pearsonothuria graeffei. J. Sci. Food Agric., 92, 965-974.
•  Zhong,  Y.,  Khan,  M.A., and  Shahidi,  F. (2007). Compositional characteristics and antioxidant properties of fresh and processed sea cucumber (Cucumaria frondosa). J. Agric. Food Chem., 55, 1188-1192.
•  Zhu,  X.,  Zhang,  K.,  Wang,  Q.,  Chen,  S.,  Gou,  Y.,  Cui,  Y., and  Li,  Q. (2015). Cisplatin-mediated c-myc overexpression and cytochrome c (cyt c) release result in the up-regulation of the death receptors DR4 and DR5 and the activation of caspase 3 and caspase 9, likely responsible for the TRAIL-sensitizing effect of cisplatin. Med. Oncol. Northwood Lond. Engl., 32, 588.
•  Ziemke,  F. and  Mantzoros,  C.S. (2010). Adiponectin in insulin resistance: lessons from translational research. Am. J. Clin. Nutr., 91, 258S-261S.