Protective Effect Of Pu-Ren-Dan Formula On INS-1Cell Injury Induced By Glucolipotoxicity And Its Mechanism

Background:Type2diabetes mellitus (T2DM) is a chronic disease that could result disability and shortened life expectancy. Abnormal reduced cell mass and dysfunction of insulin secretion of pancreatic islet β cell are the key causes of type2diabetes mellitus. Although glucose and fat are essential nutritional elements of organisms, high level of them for long-term might cause apoptosis of β cells and decline of insulin secretion, and will ultimately induce type2diabetes mellitus. Insulin signaling pathways in islet β cell have regulation effects on β cell mass and insulin secretion, which play an important role in glucolipid metabolism.Objective:To explore the injury effect of high glucose and palmitic acid on islet β cell, and confirm the protect effect of Pu-Ren-Dan formula (PRD) on injured β cell by investigatin cell viability, apoptosis and insulin secretion. And indicate the effect of PRD on insulin signaling pathways in islet β cell from molecule level, to clarify and the potential mechanisms of protective effect of PRD on islet β cells and provide experimental evidences for clinical application of PRD.Methods:INS-1cell line was used in this in vitro study, and cultured in33.3mM glucose,0.5mM palmitic acid or both33.3mM glucose and0.5mM palmitic acid to establish the gluco-and lipo-toxicity models. INS-1cell of each model group was treated with PRD serum (5%and10%) and10%metformin (MF) serum for24hours, respectively. Then CCK-8assay was used to measure cell viability; Hoechst33342staining, Flow Cytometry, AnnexinV-FITC/PI staning and caspase-3,-8Activity Assay Kit were deployed to detect INS-1cell apoptosis; and insulin secretion test was performed to evaluate INS-1cell function. And western blotting assay was deployed to investigate protein expressions of IRS2, Akt, BAD, FOXO1, mTOR, P70s6k and PTP1B, as well as the phosphorylations of IRS2(ser731), Akt (thr308), BAD (ser136), FOXO1(ser256), mTOR (ser2448) and P70s6k (thr389).Results:1. Compare of cell mass and insulin secretion among three injury models:(1) Exposure of INS-1cell to33.3mM glucose for12hours, compared to control group, cell viability (P=0.013), basal insulin secretion (BIS, P<0.001) and glucose-stimulated insulin secretion (GSIS, P=0.046) were significantly increased; after24hours, cell viability and insulin secretion showed decreasing time-independently; meantime, apoptosis was observed markedly (P<0.001).(2) Exposure of INS-1cell to0.5mM palmitic acid for6hours, cell viability was decreased, while BIS was lightly increased with no significant difference (P>0.05); after24hours, cell viability (P=0.001) and GSIS (P<0.001) were significantly reduced, but BIS and apoptosis were increased obviously (P<0.001).(3) Exposure of INS-1cell to0.5mM palmitic acid and33.3mM glucose simultaneously for6hours, cell viability (P=0.041) and GSIS (P<0.001) were significantly and time-independently decreased; after12hours, BIS also showed decreasing markedly (P<0.001), and apoptosis rate was raised, and higher than other two cell models.2. Protective effect of PRD on injured INS-1cell:High glucose, palmitic acid and high glucose with palmitic acid reduced cell viability significantly (P<0.001), but high-dose PRD could inhibit this reduction, and increased it markedly (P=0.003or P<0.001) while low-dose PRD had no significant effect on INS-1cell viability, which was similar to positive drug MF. High glucose, palmitic acid and high glucose with palmitic acid promoted cell apoptosis (P<0.001), and raised activities of caspase-3and caspase-8(P<0.001). Low-dose PRD had no significant effect on INS-1cell apoptosis, but high-dose PRD reduced activities of caspase-3and caspase-8(P<0.001), and apoptosis was inhibited (P<0.001), which was similar to the effects of MF. High glucose, high glucose with palmitic acid decreased INS-1cell BIS and GSIS (P<0.001), while palmitic acid reduced GSIS (P<0.001) but increased BIS (P=0.012). Low-dose PRD had no significant effect on INS-1cell insulin secretion, but high-dose PRD significantly increased INS-1cell BIS and GSIS (P<0.001), except for the BIS of palmitic acid group (P=0.099). The effect of PRD on INS-1cell insulin secretion was similar to MF.3. High glucose, palmitic acid and high glucose with palmitic acid significantly down-regulated protein expression of IRS2(P<0.001); reduced phosphorylations of Akt (thr308), BAD (ser136), FOXO1(ser256), mTOR (ser2448) and P70s6k (thr389)(P<0.001); up-regulated protein expressions of FOXO1and PTP1B (P<0.001); increased phosphorylations of IRS2(ser731)(P<0.001). After PRD treatment for24hours, protein expression of IRS2, phosphorylations of Akt (thr308), BAD (serl36), FOXO1(ser256), mTOR (ser2448) and P70s6k (thr389) were increased (P<0.001), while protein expressions of FOXO1and PTP1B, and phosphorylations of IRS2(ser731) were reduced significantly (P<0.001).Conclusion:1. High glucose promoted (3cell proliferation and insulin secretion in short-term, but increased apoptosis, inhibit cell viability and insulin secretion in long-term of high glucose. The influence of high glucose on GSIS was more significant than BIS; palmitic acid inhibited (3cell viability and promoted apoptosis and BIS, and GSIS was increased by palmitic acid in short-term, but decreased in long-term; palmitic acid with high glucose inhibited (3cell viability, decreased insulin secretion and promoted apoptosis, and the effects of palmitic acid with high glucose were stronger than that of palmitic acid or high glucose.2. PRD as an effective antidiabetic Chinese medicine formula could protect islet β cell against gluco-and lipo-toxicity induced injury by inhibiting apoptosis, raising cell viability, reducing activities of caspase-3and-8, and promoting insulin secretion.3. PRD inhibited the abnormal degradation of IRS2and its serine phosphorylation, which ensured the adequate IRS2proteins and its tyrosine phosphorylation, and alleviated the inhibition of Akt activity by glucolipotoxicity, promoted threonine phosphorylation at thr308to activate the PI3K/Akt pathway, then the downstreat effector proteins BAD, FOXO1, mTORand P70s6k were activated to conduct their biological functions to inhibit P cells apoptosis, promote cell viability, and improve insulin secretion.