Protective effect of nobiletin on isolated human islets survival and function against hypoxia and oxidative stress-induced apoptosis

Islets transplantation, as a treatment of type 1 diabetes, faces challenges, including the loss of islets in the process of isolation and pre-transplantation due to cellular stresses-induced apoptosis. Accordingly, the optimization of culture plays a decisive role in the transplantation success. In this study, we evaluated the effect of nobiletin on the cultured human islets. Isolated human islets were treated by different concentrations of nobiletin and cultured for 24 and 72 hours. Then, the islets viability, apoptosis, insulin and C-peptide secretion, and apoptosis markers were evaluated. Also, the production of reactive oxygen species (ROS), hypoxia inducible factor 1 alpha (HIF-1α), and its target genes in the islets were examined. Our findings showed that the islets were encountered with hypoxia and oxidative stress after isolation and during culture. These insults induced apoptosis and reduced viability during culture period. Moreover, the secretion of insulin and C-peptide decreased. Nobiletin treatments significantly improved the islets survival through reduction of HIF-1α and ROS production and suppression of apoptosis, along with increased islets function. Islet protective effect of nobiletin might be related to its anti-oxidant, anti-apoptotic and insulinotropic properties. Hence, in order to achieve viable and functional islets for clinical transplantation, the application of nobiletin during pre-transplantation period is useful.


Results
Nobiletin ameliorated islet survival in culture. The evaluation of islets showed that the percentage of viable islets was nearly 100% in the treatment and control groups at 24 hours, while the viability rate was reduced to fewer than 50% in the control group during 72 hours. Treatment with nobiletin increased the viable islets, which was significant in 10 µM concentration (Fig. 1). Thus, the percentage of recovery was about 50-60%.
Nobiletin reduced islets apoptosis in culture. Apoptotic cells were detected by TUNEL assay based on DNA fragmentation within the islets. Reduction of apoptotic cells was revealed in the presence of nobiletin in different groups during 24 and 72 hours culture. However, this decrement was significant only in 10 μM nobiletin in comparison with the control group at 72 hours (Fig. 2).
Nobiletin changed the anti-apoptotic and pro-apoptotic molecules expression in the cultured islets. To confirm the effect of nobiletin on the islets survival, we investigated Bcl-2, Bax, and active Caspase-3 protein expression during 24 and 72 hours by immunocytochemistry and the calculated H-scores were shown as histograms (Figs 3 and 4A-C). Bcl-2 as an anti-apoptotic molecule, Bax as pro-apoptotic molecule, and active caspase-3 as an enzyme in the final stages of apoptosis were measured. Nobiletin treatment in a dose-dependent manner increased Bcl-2 protein, while it decreased Bax and active caspase-3 protein during culture periods (Fig. 3). The expression of Bax protein significantly reduced in 10 µM concentration (Fig. 4A) and the Bcl-2 level significantly increased in 5 µM and 10 µM concentrations at 72 hours treatment (Fig. 4B). Active Caspase-3 had no significant changes in 24 hours compared to the control group; however, during 72 hours culture the active Caspase-3 was increased in the control group and nobiletin treatment dose-dependently reduced the active caspase-3 level, indicating the inhibition of apoptosis. Reduction of Caspase-3 was significant in 5 µM and 10 µM nobiletin concentrations (Fig. 4C).
The gene expression evaluation of Bcl-2, Bax, and Caspase-3 indicated an up-regulated Bcl-2 mRNA and a down-regulation of Bax and caspase-3 mRNA during 24 and 72 hours culture in nobiletin treatment groups compared to the control group. The reduction of Bax was statistically significant in 5 µM and 10 µM, and enhancement of Bcl-2 was significant in 10 µM concentration (Fig. 4D,E). These results showed that nobiletin could rescue the islets from apoptosis and increase their viability.
Nobiletin increased C-peptide and insulin secretion in the cultured islets. Glucose stimulation C-peptide and insulin indexes showed no significant change in the control and nobiletin treatment groups at 24 hours. However, nobiletin treatment enhanced the C-peptide and insulin indexes compared to the control group during 72 hours incubation. This increase was significant in 10 µM concentration. The mentioned result indicated that nobiletin could improve the islet function in the culture. Furthermore, the evaluation of insulin mRNA showed that nobiletin increased the mRNA level of insulin. This increase correlated positively with up-regulation of insulin secretion. Increase of insulin mRNA level was significant in 5 µM and 10 µM of nobiletin (Fig. 5).
Nobiletin changed HIF-1α gene and protein expression in the cultured islets. In order to evaluate the hypoxic stress in the cultured islet, we detected the expression of HIF-1α as the main hypoxic marker by immunocytochemistry. It was found that the HIF-1α was expressed in the cultured islets. There was no difference in HIF-1α protein level between the control and nobiletin treated groups at 24 hours, whereas nobiletin reduced HIF-1α protein in the islets during 72 hours culture. Reduction of HIF-1α was significant at 5 µM and 10 µM concentrations. The gene expression evaluation of HIF-1α did not apparently change in any groups in the culture Nobiletin enhanced the VEGF secretion and gene expression in the cultured islets. After confirming the hypoxic stress introduced into the cultured islets, we measured VEGF as a downstream target gene of HIF-1α that was expressed in mild hypoxic condition to adopt the cells with low oxygen. Our results showed that VEGF increased in mRNA and protein secretion level in the islets probably following the HIF-1α expression. However, there was a reduction in the control group at 72 hours; nobiletin treatment dose-dependently enhanced the amount of mRNA and protein secretion level of VEGF. The increase was significant in 10 µM concentration. There was, however, no apparent change between the control and nobiletin treatment groups during 24 hours incubation ( Fig. 9).

Discussion
Protection of the isolated islets and improvement of their function are necessary for successful transplantation. The widespread loss of islets during isolation and following that in the culture period is due to several insults including hypoxia and oxidative stress that can induce apoptosis. Therefore, the study of apoptotic induced mechanisms and strategies to block or diminish this process is essential for successful islet transplantation 29 .
In this study, we used nobiletin as a pharmacological approach to mitigate human pancreatic islet apoptosis and improve the viability and functionality of them in the culture. It has been reported that nobiletin exerts www.nature.com/scientificreports www.nature.com/scientificreports/ anti-apoptotic effects through suppression of intrinsic apoptotic pathway in acute kidney injury and neurodegenerative diseases 21,22 . Our result showed that nobiletin at 10 µM concentration significantly improved the islet survival. Furthermore, during 72 hours, nobiletin inhibited the Bax and caspase-3 and increased the Bcl-2 expression at both mRNA and protein levels. These findings indicated that the dead of islets through apoptosis reduced by nobiletin treatment.
Our observations were in agreement with other studies on nobiletin. It was shown that nobiletin improved the cisplatin-induced kidney injury through decreasing Bax and Caspase-3 and increasing Bcl-2 expression 21 . Similarly, it was reported that nobiletin protected against ischemic cerebral injury 22 and improved isoflurane-induced cognitive impairment 23 in rats via up-regulation of Bcl-2 expression and activation of Akt,CREB and BDNF pathways. Since the islets show some of the characteristics of neuronal phenotype due to having an endodermal lineage 30 , the suggestive protection mechanism of nobiletin may be through Akt/CREB pathway that should be investigated on the islets.
In the next step, we surveyed the effect of nobiletin on the β-cell function. It was found that nobiletin increased the insulin at both mRNA and protein secretion levels and C-peptide secretion at 72 hours. The anti-apoptotic and insulinotopic features of nobiletin on the human isolated islets are similar to glucagon-like peptide-1and prolactin 31,32 where both these drugs reduced the number of apoptotic islets via inhibition of active Caspase-8, -9, and -3 and up-regulation of Bcl-2 in the cultured rat islets and improved the function of islets through increase in the insulin secretion 31,32 . In a recent report, nobiletin (10 µM) also inhibited β-cell apoptosis through activation of cyclic adenosine monophosphate (cAMP) pathway and downstream target protein kinase A (PKA) in INS-1D β-cell line and enhanced insulin secretion through another downstream target of cAMP called exchange proteins directly activated by cAMP (EPACs) 24 . Of note to this study, nobiletin may act via cAMP pathway in the isolated islets. The percentage of TUNEL positive cells was also calculated and shown as graphs. The experiment was done in triplicate from four donors and data were analyzed by Kruskal-Wallis and Post-hoc tests. Results were represented as mean ± SD. *p < 0.05, and ***p < 0.001.
Islets are faced with hypoxia during the isolation and pre-transplant culture period because of the disconnection from the vascular network 13 . During short-term hypoxia, HIF-1α induces the expression of genes needed for metabolic adaptation including VEGF, thereby moving toward cell survival; however, in long-term hypoxia, over-aggregation of HIF-1α induces the expression of pro-apoptotic gene Bcl-2 interacting protein 3(BNIP3) or stabilizes the tumor suppressor gene p53, and moves toward apoptosis 6,7,13,33 .
Our observation showed that nobiletin (10 µM (significantly decreased the level of HIF-1α protein, scavenged ROS production, and suppressed p53 at 72 hours. The amount of VEGF mRNA and secretion also significantly increased in the presence of nobiletin. In the hypoxic response system, the VEGF expression is upregulated not only by HIF-1-mediated increase in gene transcription, but also via increased stability of VEGF mRNA, and increased VEGF protein export 29,34,35 . In our study, the HIF-1α was decreased by nobiletin at 72 hours; however, the increase in VEGF protein was remained. With this in mind, there could be other pathways involved in nobiletin induced increase in VEGF besides HIF-1α. In the current study, it was confirmed that the islets after isolation encountered hypoxia and oxidative stress that continued in the culture period. The stabilization of HIF-1α protein following hypoxia induced the expression of VEGF mRNA that could improve the islets survival during 24 hours culture because VEGF is an important growth factor for revascularization of the islets, especially after transplantation 29 . Our observations were similar to the effect of liraglutide on the isolated rat islets where this drug promoted the islet survival via enhancement of VEGF expression by activation of mammalian target www.nature.com/scientificreports www.nature.com/scientificreports/ of rapamycin (mTOR) pathway 36 , suggesting perhaps nobiletin was acting through this important pathway in angiogenesis.
At 72 hours culture, intracellular hypoxic in the islets led to chronic aggregation of HIF-1 α and stabilization of p53 that shifted the cells toward mitochondrial apoptosis pathway. It has been reported that the aggregation of HIF-1α protein is considered as an indicator of hypoxia that correlated with islet apoptosis 6,29,37,38 . According to our study, it seems that the duration of hypoxia and amount of HIF-1α protein affects the islet survival and death that would be confirmed with HIF-1α knockdown during different culture times.
Hypoxia also induces oxidative stress in the cells through overproduction of ROS 8,10 . Previous studies have shown that overproduction of ROS in the isolated islet can lead to β-cell death, due to weak intrinsic anti-oxidant defense system 7 . Moreover, overproduction of ROS influences cells through two pathways; one of them is the stabilization of HIF-1α and p53 and another one is disruption of the mitochondrial membrane and cytochrome C release with subsequent caspase 8, 9 and 3 activation 39 . It has been revealed that nobiletin plays an important role in apoptosis inhibition by restoring anti-oxidant activity and reducing the production of ROS in acute kidney injury and CNS ischemia [21][22][23] . Thus, nobiletin might reduce the islet apoptosis through its anti-oxidant properties. The anti-oxidant feature of nobiletin on the islets was similar to exendin-4 and glutathione ethyl ester where both these drugs decreased the islet apoptosis through inhibition of ROS overproduction 7,40 .
The clinical significance of this finding is that nobiletin was able to maintain the survival of islets during 72 hours. Indeed, nobiletin helped the islets to overcome the prolonged hypoxia and oxidative stresses in the culture and in this way reduced the islets death by more than 50% and increased the insulin production and www.nature.com/scientificreports www.nature.com/scientificreports/  secretion. The effect of nobiletin on the isolated human islets was mediated via anti-apoptotic, insulinotropic, and anti-oxidant properties (Fig. 10). However, it would be important to investigate the capacity of the treated islets with nobiletin in diabetic animal models. Nowadays, most transplant centers keep the islets in the culture media for 24-72 hours before transplantation that allows additional time to control the quality of the islets, islet transportation to other centers, patient preparation, coordination of transplantation team, and preoperative immunosuppressive drug administration 14,15,41 . Accordingly, nobiletin is suggested as an appropriate pharmacological substance for protection of the islets in the pre-transplant culture period that will result in a large number of islets for transplantation, thereby improving the transplant outcomes.

Conclusions
Developing protective strategies in order to overcome stress-induced apoptosis in the isolated islets would be helpful and provide higher numbers of viable and functional islets in the pre-transplant culture period, ultimately leading to successful transplantation. Nobiletin, as a cytoprotective agent, can provide an opportunity to improve the survival and function of the human pancreatic islets through its anti-apoptotic, anti-oxidant, and insulinotropic properties.  www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/

Materials and Methods
Human islet isolation and in vitro experiment. Human islets were isolated based on the semi-automated protocol of Ricordi et al. 42 ,with a few modifications 43 . After obtaining the written informed consent for research from dead brain donors' relatives, four human pancreases (Table 1) were taken in accordance with the institutional ethics committee (IR.TUMS.REC.1394.1306). Briefly, the pancreatic duct was distended with collagenase NB1 and neutral protease (Serva, Germany). After mechanical and enzymatic digestion in a Ricordi chamber, the islets purification was performed in a continuous Biocoll (Biochrom, Germany) gradient in a cell sorter processor called COBE 2991. The counting and purity of the islet were assessed by dithizone staining and the number of islets was expressed in the islet equivalents (IEQ) 44 . The isolated islets with more than 80% purity were used to study. They were maintained in CMRL 1066 (Gibco, UK) medium containing 1% FBS (Gibco, UK), 1% antibiotic/antimycotic (Sigma, Germany), and 6.25 µg/ml ITS (Sigma, Germany) in 5% CO2 at 37 °C for a night. Then, 700 IEQ/well islets (75 IEQ/cm 2 ) were treated with nobiletin (Sigma, Germany) in three concentrations of 0.5, 5, and 10 µM [45][46][47] and incubated in 5% CO 2 at 37 °C for 24 and 72 hours. The islets without nobiletin treatment were used as the control group. The experiments were analyzed in triplicate.

Islets viability evaluation.
Viability of the islets was evaluated by live/dead fluorescent dyes method based on the combination of 5 mg/ml Fluorescein diacetate by live cells (Sigma, Germany) and 2 mg/ml propidium iodide by dead cells (Sigma, Germany). Images were captured under fluorescence microscope (Olympus, CKX53, Japan). The viability rate was calculated by the percentage of the fluorescein diacetate stained area to the total area. Islets apoptosis detection. Apoptotic islets were determined by Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using Click-iT ® Plus TUNEL Assay kit (Life technology, France) according to the manufacturer's protocol. Nuclear counter staining was done with DAPI (Sigma, Germany). Images were captured under fluorescence microscope. The ratio between the apoptotic cells and nucleuses was used to calculate the percentage of apoptotic islets.

Glucose-stimulated insulin and C-peptide secretion.
After the nobiletin treatment period, the islets were washed with PBS and RPMI 1640 without glucose (Gibco, Germany) containing 0.5% BSA, and 2.8 or 20 mM glucose was then added, and incubated in 5% CO 2 at 37 °C for 2 hours. Both supernatants were collected and stored at −80 °C. The secreted insulin and C-peptide [49][50][51][52] were measured with ELISA kit (Monobind, USA). In each group, the stimulation index was calculated by dividing the amount of C-peptide and the insulin released