Emulsified isoflurane alleviates rat islet beta cell RIN-m5F apoptosis induced by glucose via inhibiting endoplasmic reticulum stress

Diabetes mellitus (DM) is a critical disease that considered a detriment to the health of people all over the world. Endoplasmic reticulum stress (ERS) is the response cause by endoplasmic reticulum misfolded and unfolded protein aggregation, which induces cell apoptosis. Our previous work showed that EIso could alleviate ERS in lung reperfusion injury. This study aimed to elucidate whether Emulsified isoflurane (EIso) could alleviate apoptosis induced by glucose in rat islet beta cell RIN-m5F via inhibiting ERS. the expression of Bax and Bcl-2 were detected by Western The level of caspase-3 activity was by Finally, the and expression were detected The and

expression of CHOP and GRP78 were inhibited by EIso at 24 h after treatment, and decrement of CHOP and GRP78 expression were correlated with EIso concentration.
The level of ATF6, Xbp1 and eIF2α mRNA of RIN-m5F were enhanced culture with high glucose, but only eIF2α mRNA was decreased by EIso treatment.

Conclusion
High glucose induces rat islet beta cell RIN-m5F apoptosis and aggravates the function of beta cells. EIso protects beta cells from glucose-induced apoptosis, and anti-apoptosis is mediated, at least in part, by inhibiting ERS.

Background
Diabetes mellitus (DM) are series of diseases feature with hyperglycemia; the most common forms of DM are type 1 DM (T1DM) and type 2 DM (T2DM). In China in 2013, the estimated overall prevalence of DM among adults was 10.9% and that of prediabetes was 35.7% [1]. Seriously, result of obesity, unhealthy diet, sedentary lifestyle and genetic factors, the prevalence of T2DM in adolescents and young adults has been dramatically increasing [2].
In T2DM patients, during insulin resistance and impending diabetes, the pancreatic beta cell capable to synthesize and secrete sufficiently insulin for glucostasis [3].
Regretfully, excessive synthesis of insulin trigger multiple pathological reactions [4][5][6], which cause progressive loss of beta cell function, insulin deficiency and overt T2DM if there are no effective interventions. Endoplasmic reticulum stress (ERS) is a adaptive response during stress, in T2DM patients it can be triggered by an imbalance between the folding capacity of the Endoplasmic reticulum (ER) and irreparably unfolded and misfolded proteins aggregate in lumen cause by excessive pro-insulin synthesis [7]. Recent studies authenticated that ERS involved in the development of insulin resistance and progression to T2DM, and inhibit ERS would protect the function of beta cells [7,8].
Emulsified isoflurane (EIso), emulsified preparation of isoflurane, has marked anesthetic potency and comparable safety index and certain safety factor as propofol in patients and animals [9,10]. Furthermore, researches demonstrated that EIso had extensive protection in organs, including liver, lung and kidney [11,12].
Whether EIso could protect pancreas remains unknown. Our previous study showed that EIso administered before ischemia could protect lung against reperfusion injury through inhibiting ERS pathway (published in Chinese). Therefore, we hypothesized that EIso could contribute to increasing beta cell mass by inhibiting ERS.

Cell Culture and treatment
Rat islet beta cell line (RIN-m5F) was purchased from China Center for Type Culture Collection (CCTCC). The cells were cultured in RPMI-1640 (Gibco, USA) containing 10% fetal bovine serum (Gibco, USA) at 37℃ and a 5% CO 2 atmosphere. RIN-m5F cells were then randomly divided into five doses of glucose and EIso for different culture. EIso (8%) was obtained from the Laboratory of Anesthesiology and Critical Care Medicine, West China Hospital of Sichuan University (Chengdu, China). On attaining 70-80% confluency, cells were seeded into 6-wellplate at a density of 2 × 10 6 and grown over night. We choose glucose with final concentrations of 0.1M (0.1G group) and 0.3M (0.3G group) to research the influence of high glucose to RIN-m5F cells. We use 57uM (0.3G+57E group) and 76uM (0.3G+76E group) EIso to study the EIso-protection based on preliminary experiments.

Cell viability assay
Indicated cells were cultured in 96-well plates, followed by drug exposure for 24h.The rate of cell apoptosis was examined by MTT assay. Briefly, after exposure to glucose and EIso, 200ul of MTT solution (0.5mg/ml, Sangon Biotech, Shanghai, China) was subsequently added. After 4h, 100 ul of dimethyl sulfoxide was added and mix gently for 10minutes. Then, measure the absorbance at 570 nm using microplate reader.

Insulin level assay
Cells were seeded overnight in 6-well plates and treated as indicated. Insulin levels in culture medium were quantified using an insulin rat ELISA kit ((Beyotime Biotechnology, Nantong, China) according to the manufacturer's instructions.

Caspase-3 activity assay
Caspase-3 activity was measured using the Caspase-3 Activity Assay kit (Beyotime Biotechnology, Nantong, China) following the manufacturer's instructions. In brief, the protein samples from cells were obtained and 100 µg protein was added to a reaction buffer containing Ac-DEVD-pNA (2mM), incubated at 37˚C for 2 h. the absorbance of yellow pNA (the cleavage product) was measured with microplate reader at 405nm. The caspase-3activity was recorded as the ratio to that of the control group.

Western blotting analysis
After treatment, cellular protein was extracted using RIPA lysis buffer (Beyotime Biotechnology, Nantong, China) containing 1mM PMSF for 30 minutes on ice. The supernatant containing soluble total protein was collected after centrifuge at 12,000rpm for 10min at 4˚C. The BCA Protein Assay Kit (Sangon Biotech, Shanghai, China) was used to evaluate the protein concentrations. Approximately 50ug of protein was separated by electrophoresis in 12% SDS-polyacrylamide gels, and were subsequently transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat milk powder in 0.01M PBS (pH 7.4) and 0.05% Tween-20 at room temperature for 1h. Subsequently, they were incubated with primary antibodies against Bax, Bcl-2, GPR78, CHOP (Absin Bioscience, Shanghai, China,

EIso improves the survival and protects the function of RIN-m5F
We evaluated the influence of high glucose on the morphology in RIN-m5F cells, after exposed to 0.1M and 0.3M glucose for 24 hours, cell shrinkage appeared, quantity and rate of cellular attachment were reduced. Cells shrinkage and rounded off were significantly decreased, quantity and rate of cellular attachment were significantly increased when 57uM or 76uM EIso was added (Fig.1A).
In addition, increasing of glucose in the culture media induced significant loss of cell viability, as estimated by the MTT assay, and in a concentration-dependent manner. Compare to control group, the viability significantly decreased in 0.

Discussion
Glucotoxicity, which refers to irreversible damage of islet beta cells, plays an important role in the development of DM [13]. There are lots of articles involved in glucose induced apoptosis about rodent primary islet cells, INS-1 cells and MIN6 cells [14,15]. But few articles pay attention to rat islet beta cell RIN-m5F. Thus, in this study, we validated the damage of glucose on RIN-m5F. Our results showed that 0.1M and 0.3M glucose stimulate the secretion of insulin, alter the morphology and decrease the viability within 24h treatment. Kornelius et al. also revealed that exposure of RIN-m5F cells to high glucose and FFA levels increase apoptosis [16].
The effect of glucotoxicity on RIN-m5F was explicit, we next to sought the protection against glucotoxicity.
Isoflurane is an isomeride of enflurane, which was commonly used in inhalation anesthesia. But, isoflurane can induce neurogenetic damage and neurocognitive disorder, even accelerate the process of Alzheimer's disease [17][18][19].With a view to the adverse effects of inhalational anesthetic compare to intravenous anesthetic.
EIso was manufactured by West China Hospital of Sichuan University, which liquid isoflurane was mixed with intralipid at the final concentration of 8% (v/v). Except marked anesthetic potency and safety, Hu et al. demonstrated that EIso pretreatment protects the myocardial ischemia and reperfusion injury in rats, which may mediated by the inhibition of apoptosis and cell damage [20]. In this study, we found that EIso protects the morphology and enhances the viability of RIN-m5F and restores the synthesis of insulin in 0.1M and 0.3M glucose treatment. Besides, our results showed EIso inhibits the activity of caspase-3. Caspase-3 is a predominant player in the execution of apoptosis, which can target key structural and regulatory proteins to bring about apoptotic cell death [21]. Cao et al. indicated that Caspase-3 is critical for the induction of MIN6 cells apoptosis and it's activation is further confirmed to be related to the NF-κB-mediated Bcl-2 down-regulation [22]. B cell leukemia/lymphoma 2 (Bcl-2) family proteins are the key to the regulation and execution of apoptosis, among them, Bcl-2 is a pro-survival protein, and Bcl2 associated X (Bax) is a pro-apoptotic protein [23]. In this study, we found that high glucose induced Bcl-2 expression decrease and increase Bax expression, but EIso down-regulated the expression of Bax and up-regulated the expression of Bcl-2.It seems EIso protected RIN-m5F against glucotoxicity may relevant to apoptosis.
The mechanisms of apoptosis are highly complex, involving a cascade of molecular events. Increased protein synthesis, imbalance of ER calcium levels, glucose and energy deprivation that trigger ERS can induce beta cell failure by UPR [24]. Glucose regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) are the best studied inducer of ERS. GRP78 is an important sensor of misfolded proteins in ER lumen. When misfolded proteins accumulated, GRP78 was competed binding by misfolded proteins with EIF2AK3, which resulted in protein kinase RNA (PKR)-like ER kinase (PERK) activation and phosphorylation of eukaryotic translation initiation factor-2 (eIF2a) [25]. A increased availability of GRP78 may re-establishment homeostasis of endoplasmic reticulum by attenuating UPR [26]. UPR protects cells from stress and contributes to the re-establishment of endoplasmic reticulum homeostasis; however, prolonged UPR promotes CHOP expression. CHOP induces the expression of growth arrest and DNA damage 34 (GADD34), activates death receptor in 57uM and 76uM. ERS trigger downstream response mediated by three canonical signaling sensors: protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring enzyme 1α (IRE1α), and activating transcription factor-6 (ATF6) [28]. Next, we detected the mRNA expression of ATF6, X-box-binding protein 1 (Xbp1) and eIF2α.
Xbp1 is a molecule that mediated with the activation of IRE1α. Our results show that high glucose increased the expression of ATF6, Xbp1 and eIF2α, and eIF2α mRNA expression decreased after treated with 57uM and 76uM EIso. These results provide the evidence that EIso protects RIN-m5F may via, at least in part, inhibiting ERS.

Conclusion
The current study demonstrated ERS-mediated apoptosis was induced by high glucose in rat islet beta cell RIN-m5F. EIso promoted RIN-m5F function and antiapoptosis by inhibiting ERS. The application of EIso during operation of DM patients may protect the function of pancreas and impede the stress hyperglycemia.

Declarations
Ethics and Consent to Participate Not Applicable.

Consent to publish
Not applicable.

Availability of data and materials
The datasets generated during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.