MuhammaD Shahzad
Age 17 | Edmonton, AB
Canada-Wide Science Fair 2019 Excellence Award: Senior Gold Medal & Canadian Young Researcher Special Award | Canadian National Chemistry Special Award 2019 | Edmonton Regional Science Fair 2019 Grand Award
Type 1 diabetes mellitus (T1DM) is an autoimmune disease that accounts for 10-15% of all diagnosed cases of diabetes and usually occurs in childhood or young adulthood (Atkinson et. al 2013). T1DM is characterized by the autoimmune destruction of insulin-secreting β cells located within islets of Langerhans — regions of the pancreas that contain its endocrine cells (Atkinson et al. 2014). T1DM may result in secondary complications such as cardiovascular disease, neuropathy, nephropathy, and retinopathy. Some of these complications may be life-threatening even when blood glucose levels are well controlled. A potential treatment option for T1DM is islet transplantation where islets of Langerhans are isolated from a donor pancreas and transplanted into patients (Shapiro et al. 2006).
Introduction
During the past 25 years, considerable progress has been achieved in clinical islet transplantation, especially after the development of the Edmonton Protocol (Shapiro et al. 2006). The Edmonton Protocol allows for islet transplantations to be conducted in a minimally invasive manner, whereby donor islets are injected into the patient intravenously and settle in the portal vein of the liver. A challenge facing its wider implementation is the shortage of donor islet tissue for transplantation (Shapiro 2013). Furthermore, islets undergo stress during isolation procedures that separate them from the donor pancreas prior to transplantation and consequently become damaged or undergo apoptosis (a form of regulated cell death), worsening existing issues of islet tissue scarcity (Bottino et al. 2006, Evgenov et al. 2006, Emamaulle & Shapiro 2007).
A particular type of stress that is a major cause of islet death during isolation is oxidative stress, which is caused by highly reactive derivatives of molecular oxygen called reactive oxygen species (ROS) (Hennige et al. 2000, Bottino et al. 2004). Examples of ROS include the superoxide radical and hydrogen peroxide. Islets and β cells are prone to oxidative stress-induced injury because of their inherently low levels of antioxidant enzymes superoxide dismutase 1 (SOD-1), glutathione peroxidase 1 (GPX-1), and catalase (Tiedge et al. 1997, Robertson 2006). These enzymes are critical components of cellular defense mechanisms against the harmful effects of oxidative stress. (Birben et al. 2012). Exposure to high levels of ROS, which are not detoxified by cellular antioxidants, results in oxidative stress and ultimately cell death. It is well documented that oxidative stress contributes to cell injury during islet isolation and transplantation protocols (Kajimoto & Kaneto 2004).
The focus of this project involves investigating if Pomelo (Citrus grandis) and Chinese bayberry (Myrica rubra) fruit extracts can protect mouse β cells from the harmful effects of oxidative stress. These extracts have been shown previously to be composed of a rich mixture of antioxidant molecule. They are well-established to have more potent antioxidant properties than other fruits, so treating cells with these extracts may protect them from ROS-induced oxidative stress (Zhang et al. 2013, Caengprasath et. al 2012). As such, a potential solution to the issue of islet death during isolation may be to treat donor cells and tissue with these extracts, which may improve cell viability and islet yield by partially protecting the cells from oxidative stress.
The hypothesis for this project was that Pomelo and Chinese Bayberry fruit extracts will protect mouse islet β cells from the harmful effects of oxidative stress induced by ROS because both of these extracts have previously demonstrated potent antioxidant properties (Zhang et. Al 2011 and Caengprasath et. Al 2013).
MATERIALS & METHODS
Cell Line
Mouse beta TC-tet cell line was used in this project. The rationale for using this immortalized mouse cell line was that it is cost effective and because it is a common cell line used for type 1 diabetes research.
Cytotoxicity Assay
The toxicity of Pomelo and Chinese Bayberry, as well as the viability of cells treated with or without either of the extracts prior to ROS exposure was determined using the Trypan Blue exclusion dye method. β cells were cultured in Dulbecco’s modified eagle medium (DMEM) with or without Pomelo or Chinese Bayberry extracts (0.5 mg/mL) for 22 hours prior to exposure to H₂O₂ for 2 hours. A dose-dependent response element was completed before this project that found 0.5 mg/mL concentration of both extracts was non-toxic to cells. All incubations were conducted at physiological conditions (37°C, 5% CO2, 95% air). Untreated cells served as controls. After culture, the cells were collected and stained with Trypan blue. The percentage of live cells was determined by counting the live and dead cells using hemocytometer and light microscope.
Western Blot Analysis
A western blot experiment was conducted to analyze protein expression of SOD-1, GPX-1 and catalase enzymes in β cells. β cells were treated with or without Pomelo or Chinese Bayberry extracts (0.5 mg/mL) prior to H₂O₂ exposure. Untreated cells served as control. After treatment, total cell proteins were extracted using RIPA lysis buffer and cytoplasmic proteins were extracted using NE-PER cytoplasmic extraction reagents (Thermo Scientific) according to manufacturer’s protocol. Western blot was performed as previously described with few modifications (Li et al. 2015). Briefly, 20 µg proteins were separated on 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride membranes (Millipore). Following a 1-hour blocking in phosphate buffer solution containing 1% Tween-20 (Sigma-Aldrich Canada Co.) and 2% BSA, the blots were incubated overnight at 4°C with the primary antibodies. The following primary antibodies and their respective dilutions were used: β-actin (1:1000, Cell Signaling Technology), SOD-1 (1:1000, Millipore), GPX-1 (1:1000, Millipore), and catalase (1:1000, Millipore). The membranes were then further incubated for 1 h with IRDye goat anti-rabbit IgG secondary antibody (1:5000, LI-COR, Lincoln, NE, USA). Finally, the bands were visualized using the Odyssey CLx infrared imaging system (LI-COR). The protein concentrations were normalized to the internal control β-actin and values were expressed as normalized data relative to the control.
Total RNA Extraction & RT-qPCR
Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to analyze gene expression of anti-apoptotic molecules BCL2 and SURVIVIN. The total RNA was extracted from mouse β cells treated with or without Pomelo or Chinese Bayberry fruit extracts prior to H2O2 exposure using Trizol reagent (Invitrogen) to evaluate expression of BCL2 and SURVIVIN transcripts. The total RNA was used to construct cDNA using Superscript RNase H-Reverse Transcriptase (Invitrogen). PCR amplification were performed on a Light Cycle Real-time PCR thermocycler (Roche Diagnostics) using Taqman gene expression assays. All gene expressions were normalized to the internal control 18S RNA.
Apoptosis Detection Assay
The number of apoptotic and necrotic cells in β cells treated with or without Pomelo or Chinese Bayberry extracts prior to H₂O₂ exposure was measured using the Annexin V-Cy3 Apoptosis detection kit. 6.0 x 106 β cells were incubated in DMEM media with or without Pomelo or Chinese Bayberry fruit extracts (0.5 mg/mL) at 37°C, 5% CO2, 95% air prior to H2O2. After incubation, the cells were collected and assayed for cell viability using Annexin V-Cy3 Apoptosis detection kit (Sigma-Aldrich Canada Co.) following the manufacturer’s protocol. This kit includes annexin V conjugated to Cy3.18 as the fluorochrome and the non-fluorescent compound 6-carboxyfluorescin diacetate (6-CFDA) that is able to readily diffuse into the cell and is hydrolyzed by the esterases in live cells to the fluorescent compound 6-carboxyfluorescein. As such, the kit allows for the differentiation among early apoptotic cells (annexin V positive, 6-CFDA positive), necrotic cells (annexin V positive, 6-CFDA negative) and viable cells (annexin V negative, 6-CFDA positive). Additionally, the beta cells were stained with 4’,6-diamidino-2-phenylindole, dihydrochloride (DAPI, Invitrogen), which stains the nucleus of the cell blue, for further identification. Images of the stained β cells were attained using the Zeiss fluorescent microscope (Carl Zeiss) and analyzed using ImageJ software. The number of live, necrotic, or apoptotic cells for each condition was counted and the number of these cells over four trials were added together and then divided by 4 to obtain the mean number of live, necrotic, or apoptotic cells under each condition. The mean percentage of live, necrotic, or apoptotic cells was then found by dividing the mean number of live, necrotic, or apoptotic cells by the mean total number of cells (live plus necrotic plus apoptotic) counted under each condition and multiplying by 100.
Table 1. The viability of β cells was determined using Annexin V-Cy3 apoptosis detection kit and analyzed using ImageJ software. Data shown in the table are averages of three independent trials. Apoptotic (Annexin V positive, 6-CFDA positive), necrotic (Annexin V positive, 6-CFDA negative), and viable cells (Annexin V negative, 6-CFDA positive) can be detected. The number of live, apoptotic, and necrotic cells under each condition were counted. The percentage of live, apoptotic, or necrotic cells was calculated by dividing the number of live, apoptotic, or necrotic cells counted by the total number of cells (live + apoptotic + necrotic) and multiplying the result by 100.
*P<0.05 H₂O₂ vs. Untreated, ¶P<0.05 H₂O₂ vs. Pomelo, ¶ ¶P<0.05 H₂O₂ vs. Chinese Bayberry, **P<0.05 Pomelo + H₂O₂ vs. H₂O₂, ∞P<0.05 Chinese Bayberry + H₂O₂ vs. H₂O₂
Statistical Analysis
All experiments were performed at least in triplicate and the data is presented as mean ± standard error of the mean (S.E.M.). Statistical analysis was performed with Stat/IC 13.0 software (Stata Corp LLC, College Station, TX, USA). Differences were considered statistically significant when P value was 0.05 or less using Student’s t-test analysis and non-parametric analysis of variance (ANOVA).
RESULTS
Pomelo and Chinese Bayberry Extracts Were Non-Toxic to β cells and Improved Viability Upon Oxidative Stress
To determine the cytotoxicity of Pomelo and Chinese Bayberry fruit extracts on β cells and if pretreatment with either of the extracts could improve β cell viability following the ROS insult, the cells were subjected to Trypan blue exclusion dye staining. The Trypan blue analysis showed that there was no statistically significant difference in viability between cells treated with either of the extracts and the untreated control (Figure 1). However, cells that were pre-treated with the extracts prior to H₂O₂ exposure had a significantly higher viability than cells that were exposed to H₂O₂ without pretreatment with the Pomelo or Chinese Bayberry extracts (Figure 1).
Figure 1. Cell viability measured by Trypan blue exclusion dye method of β cells treated with or without the Pomelo or Chinese Bayberry fruit extracts prior to H₂O₂ exposure. The cells were incubated in DMEM media with or without either of the fruit extracts at concentrations of 0.5 mg/mL at physiological conditions (37ºC, 5% CO2, 95% air After 22 hours of incubation with or without the extracts, some of the cells were exposed to 0.4 mM H₂O₂. Results are presented as means ± S.E.M. from five independent experiments. *P<0.05 vs H₂O₂, **P<0.05 vs the untreated cells.
Treatment with Pomelo & Chinese Bayberry Extracts Significantly Reduced ROS-Induced Apoptosis in β cells
The Annexin V-Cy3 cell viability assay was conducted to determine the effects of treating the β cells with Pomelo or Chinese Bayberry extracts prior to H₂O₂ exposure on the number of live, necrotic, and apoptotic cells. The assay shows that there was no significant difference in the proportion of live, necrotic, and apoptotic cells between the untreated controls and the cells treated with either Pomelo or Chinese Bayberry extract on its own, confirming my finding that Pomelo and Chinese Bayberry extracts were not toxic to mouse β cells. However, I found that the percentage of live cells in β cells treated with H₂O₂ (35.2% ± 0.2%) on its own was significantly (P<0.05) lower than the percentage of live cells in untreated β cells (91.8% ± 0.4%) and those that were pre-treated with Pomelo (92.1% ± 0.3%) or Chinese Bayberry (91.7% ± 0.5%) fruit extracts prior to H₂O₂ exposure. I also found that the percentage of apoptotic cells in β cells treated with H₂O₂ on its own was significantly higher (P<0.05) than in untreated cells as well as cells treated with Pomelo or Chinese Bayberry extracts prior to H₂O₂ exposure.
Treatment with Pomelo & Chinese Bayberry Significantly Upregulated Protein Expression of Antioxidant Enzymes
To investigate the possible mechanism of protection of the extracts on β cells, the protein expression of numerous antioxidant enzymes involved in cellular defense mechanisms against oxidative stress were analyzed using western blot. Treatment with Pomelo or Chinese Bayberry fruit extracts on their own or prior to H₂O₂ exposure significantly upregulated the protein expression of SOD-1, GPX-1, and catalase antioxidant enzymes (Figure 2A, B, C, D).
Figure 2. Protein expression of SOD-1, GPX-1, and catalase enzymes on mouse β cells treated with or without the Pomelo (A) or Chinese Bayberry (B) fruit extracts prior to H₂O₂ exposure. Bar graphs showing the band intensities relative to the levels detected in untreated β cells by densitometric quantification (C and D). Untreated controls were normalized to 1. Data are expressed as means ± S.E.M. from three independent experiments. *P<0.05 vs. the untreated β cells and ∞P<0.05 vs H₂O₂ .
Treatment with Pomelo & Chinese Bayberry Significantly Up Treatment with Pomelo & Chinese Bayberry Significantly Upregulated Gene Expression of Anti-apoptotic Molecules
To further elucidate the underlying protective mechanism of Pomelo and Chinese Bayberry extracts, I evaluated the gene expression of anti-apoptotic molecules BCL2 and SURVIVIN by RT-qPCR. Treatment of the β cells with either of the extracts (0.5 mg/mL) on their own or prior to H₂O₂ exposure significantly enhanced the BCL2 and SURVIVIN gene expressions (Figure 3A, B).
DISCUSSION
In the present study, I showed that Pomelo and Chinese Bayberry fruit extracts have a protective effect on β cells. I found that treatment of β cells with either of the extracts at 0.5 mg/mL was non-toxic, as there was no significant difference in viability between treated and untreated cells. This dose was chosen because a pre-liminary viability assay that investigated the dose-dependent effect of Pomelo and Chinese Bayberry extracts on the cells found that 0.5 mg/mL concentration was non-toxic (data not shown). I was able to reproduce this in subsequent experiments. Also, the pre-treatment of β cells with either of the extracts prior to ROS exposure significantly improved viability. Moreover, I found that significantly more live cells and less apoptotic cells were detected in β cells treated with Pomelo or Chinese Bayberry extracts prior to H₂O₂ exposure than in cells treated with H₂O₂ without pretreatment, suggesting that Pomelo and Chinese Bayberry extracts may have a protective effect on H₂O₂-induced β cell death.
SOD-1, GPX-1 and catalase are among the most critical components of cellular defense systems against oxidative stress and are thought to play a major role in maintaining antioxidant/oxidant imbalance (Maines 1997). Some studies have suggested that the induction of these antioxidant enzymes may promote graft survival when islets are transplanted under the kidney capsule in mouse models (Bottino et al. 2004, McCall & Shapiro 2012). β cells have low basal levels of these antioxidant enzymes; consequently, they are prone to ROS-induced injury and ultimately oxidative stress. My results show that β cells treated with Pomelo or Chinese Bayberry extracts had significantly higher protein expression of these antioxidant enzymes compared to untreated cells.
BCL2 and SURVIVIN are well-known molecules that inhibit cell apoptosis. Indeed, some studies have suggested that these molecules can protect pancreatic β cells and islets from apoptotic cell death (Tran et al. 2003, Dohi et al. 2006). As such, I also assessed the gene expression of BCL2 and SURVIVIN in mouse β cells. I found that the gene expression of both of these anti-apoptotic molecules was significantly upregulated in β cells treated with either Pomelo or Chinese Bayberry extracts when compared to the untreated cells. The upregulation of the gene expression of these molecules may suggest an underlying mechanism by which treatment with the extracts seemed to reduce the number of apoptotic β cells; however, it remains to be seen if protein expression correlates with gene expression.
FUTURE DIRECTIONS
Assessing the protein expression of BCL2 and SURVIVIN molecules using western blot will be completed in the near future to determine if protein expression correlates with the gene expression results of this study and to further elucidate the underlying mechanism of protection of Pomelo and Chinese Bayberry extracts on mouse β cells.
NRF2, a basic region leucine-zipper transcription factor, has been determined to be involved in the transcriptional regulation of antioxidant enzymes SOD-1, GPX-1, and catalase (Alam & Cook 2003). NRF2 contains a transcriptional domain that can positively regulate the expression of SOD-1, GPX-1 and catalase genes. When NRF2 is activated, it dissociates from its docking protein KEAP1 in the cytosol and migrates into the nucleus, where it binds to the antioxidant response element (ARE) in the promoter and thus stimulates expression of the aforementioned antioxidant genes.
It has also been suggested that phosphorylation of several signal transduction pathways including PI3K/AKT could activate NRF2 leading to the translocation of NRF2 (Zhao et al. 2015). The PI3K/AKT pathway has also been linked to the activation of the anti-apoptotic molecule BCL2 in the cytoplasm of cells. Activated PI3K phosphorylates AKT, and the phosphorylated AKT then phosphorylates the Bad protein. Bad is bound to BCL2, but when it gets phosphorylated by p-AKT, it dissociates from BCL2, releasing it into the cytoplasm and thus inhibiting cell apoptosis.
As such, to investigate the mechanism by which pre-treatment with Pomelo or Chinese Bayberry extracts upregulates antioxidant enzyme expression and prevents apoptotic cell death, I will be evaluating the expression of phosphorylated Nrf2, phosphorylated AKT, and phosphorylated Bad using western blot. I will also investigate protein-protein interaction between Bad and BCL2 in cells using co-immunoprecipitation and western blot analysis.
To investigate whether cells pre-treated with either of the fruit extracts could improve engraftment and surgery outcomes, a future direction for this project would be to conduct in vivo trials, where islets pre-treated with either Pomelo or Chinese Bayberry extracts would be transplanted in mice.
Ultimately, the results of this project support my hypothesis and suggest a potential solution to the issue of donor scarcity that is hindering the further implementation of islet transplantation to treat patients with type 1 diabetes. Since nearly 40% of islets die for every pancreas during the isolation procedures (Shapiro et. Al 2018), treating the donor pancreas and isolated islets with these fruit extracts during isolation and culturing prior to transplantation may increase the viability of the cells and thus provide an increased tissue yield for transplant. As such, the results support a potential method to alleviate the issue of donor tissue scarcity for islet transplantation and ultimately make the procedure accessible to more patients.
ACKNOWLEDGEMENTS
The author is grateful to Dr. Gina Rayat at the University of Alberta for allowing him to conduct this research project in her lab, and for her continued support. The author is also grateful for his high school science teacher Ms. Robyn Ferguson and Old Scona Academic High School (Edmonton, Alberta) for continued support in his research endeavours.
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Muhammad Shahzad
Muhammad Shahzad is a grade 12 student at Old Scona Academic and a biomedical researcher in the Faculty of Medicine at the University of Alberta. Muhammad is an avid science enthusiast, regularly competing in science competitions and organizing STEM programs for youth. He has been recognized for outstanding research at the national level and is an outspoken advocate for youth involvement in STEM. When he isn’t tinkering away in the lab, you can find Muhammad watching the latest episode of House MD or cheering on the Raptors.