Institute of Biomedical studies
Withaferin A prevents islet damage caused by IBMIR: microRNA 375 analysis
Mazhar Kanak1, Morihito Takita2, Rauf Shahbazov2, Marlon F Levy3, Michael C Lawrence2, Bashoo Naziruddin3.
1Institute of Biomedical Studies, Baylor University, Waco, TX, United States; 2Baylor Research Institute, Baylor University Medical Center, Dallas, TX, United States; 3Baylor Simmons Transplants Institute, Baylor University Medical Center, Dallas, TX, United States
Background: Islet transplantation is a promising therapy for maintaining or replacing beta cell function to patients with chronic pancreatitis and type 1 diabetes respectively. One of the major impediments that need to be addressed to improve islet transplant outcome is the Instant Blood Mediated Inflammatory Reaction (IBMIR). Previously we have reported the beneficial effect of NFκB inhibitor, Withaferin A (WA) on islets from proinflammatory cytokine induced damage. In this study, we used microRNA 375 (miR375), a biomarker for islets to further assess beta cell damage.
Methods: Human islets were isolated from cadaveric donor pancreases for research purpose. Purified human islets were mixed with autologous human blood in a heparinized tube and placed at 37 °C in a shaker incubator. For WA treatment, islets were pretreated with 1 µg/mL WA for 1hr prior to mixing with blood. Plasma samples were collected at various time points and stored at -80C until analysis. Total microRNA was isolated from plasma using exiqon biofluid miRNA isolation kit. MicroRNA were reverse transcribed and real time PCR was performed using exiqon universal cDNA synthesis kit and real time quantitative PCR kit respectively as per manufacturer’s protocol. LNA based primers for miRNA 375, miRNA 423-3p, and Unisp6 were used. Statistical significance was determined by two-way ANOVA. (Significance are denoted as *P<0.05, **P<0.01, ***P<0.001, ****P<0.00001)
Results: As reported previously, treatment of human islets with autologous human blood resulted in activation of IBMIR analyzed by markers in the serum. miRNA 375 was significantly increased in the serum immediately after mixing and the level of miR375 increased with time. Islets that were pretreated with WA had a significantly reduced miR375 levels at 1hr, 3hr and 6hr time points compared to controls. Relative expression of miR375 in serum of control vs. WA group differed at 1hr (407.26 vs. 53.99; P=0.0042), 3hr (470.9 vs. 204.77; P=0.037) and at 6hr (452.07 vs. 196.2; P=0.0462). These data correlated with the c-peptide and pro-insulin levels for the same samples.
Conclusion: Out study supported that WA may be used as a potential anti-inflammatory treatment to overcome IBMIR and miR375 is a reliable marker to evaluate islet damage.
Nofit Borenstein. Ana Rahman. Yoshiko Tamura. Baylor University Graduate Program. Baylor Health Care System.
15:30 - 17:00
|Getting Beta Cells to Survive: Beta Cell Survival and Injury Mechanisms||Withaferin A prevents islet damage caused by IBMIR: microRNA 375 analysis||Plenary Room 1|