Study On The Mechanism Of The Serum From FTZ-fed Rats On The Improvement Of Oxidative Stress In INS-1 Cell Line

BackgroundDiabetes mellitus is a metabolic disorder of fuel homoeostasis characterized by hyperglycaemia caused by early-onset peripheral insulin resistance and later β-cell dysfunction and insulin deficiency. It is regarded as one of the most important non-communicable diseases that affect global health. The harmful effect of chronic hyperglycaemia on β cells plays an important role in the development of type 2 diabetes, and this aspect has attracted significant attention. Oxidative stress resulting from exposure to high levels of glucose is considered to be one of key factors on β-cell dyfunction.It was previously reported that Fufang Zhenshu Tiaozhi Capsule(FTZ), could regulate lipid metabolism through lipid absorption, distribution, metabolism and excretion, so it could improve hyperglycaemia, hyperlipidaemia, inflammatory and protect vascular endothelium. However, it still needs further research to determine whether FTZ could protect β cells from oxidative stress induced by high glucose. Objective1. To investigate the mechanism of serum from FTZ-fed rats in the improvement of high glucose-induced oxidative stress in INS-1 cell line;2. To investigate the mechanism of serum from FTZ-fed rats in the improvement of oxidative stress-induced apoptosis in INS-1 cell line;3. To investigate the mechanism of serum from FTZ-fed rats in the improvement of glucose stimulated insulin secretion in INS-1 cell line. Methods1. Serum preparationForty healthy Sprague-Dawley(SD) male adult rats were equally distributed and divided into two groups. In the first group, each animal was orally administrated FTZ solution at a concentration of 3 g(dry FTZ powder)/kg(weight)/dose twice a day for three days. Blood was obtained through aorta abdominalis 1h after the last administration and the serum from this group was named as FTZ-serum. In the second group, rats were orally administrated water in the same protocol and the serum from this group was named as rat-serum. Both the FTZ-serum group and rat-serum group were inactivated by heating at 56℃ for 30 min, then filtered through 0.22 μm filters and stored at-80℃ until use. The FTZ preparation, FTZ-serum and rat-serum were analyzed with combined UPLC/Q-TOF-MS.2. Cell cultureINS-1 cell line was grown at 37 oC with 5% CO2 in RPMI 1640 medium. For individual experiments, cells were plated at a density of 2×105 cells/well on 35 ×10 mm culture plates and divided into control group(cultured in RPMI 1640 medium with 11 mmol/L glucose and 10% FBS), model group(cultured in RPMI 1640 medium with 25 mmol/L glucose and 10% FBS), edaravone group(cultured in RPMI 1640 medium with 25 mmol/L glucose, 10% FBS and edaravone with the concentration of 200 μmol/L), 10% rat-serum group(cultured in RPMI 1640 medium with 25mmol/L glucose and 10% rat serum), 0.4% FTZ-serum group(cultured in RPMI 1640 medium with 25 mmol/L glucose, 0.4% FTZ-serum and 9.6% rat serum), 2% FTZ-serum group(cultured in RPMI 1640 medium with 25 mmol/L glucose, 2% FTZ-serum and 8% rat serum), 10% FTZ-serum group(cultured in RPMI 1640 medium with 25 mmol/L glucose and 10% FTZ-serum). All groups were cultured lasting for 48 hours.3. Oxidative stress assaysThe levels of ROS, MDA in INS-1 cells were measured by DCFH-DA fluorescence probe and MDA detection kit respectively. The protein levels of Mn-SOD, CAT were analyzed by Western Blot.4. Apoptosis assaysThe apoptosis of each group was assayed using Annexin V-FITC kit with flow cytometry. The activity of caspase 9 and caspase 3 was detected with caspase 9 kit and caspase 3 kit respectively. The protein levels of Bcl-2, Bcl-xl, Bax were analyzed by Western Blot.5. Insulin secretion assaysThe protein levels of PDX-1, Glut 2 were analyzed by western blot. The glucose stimulated insulin secretion was analyzed with rat insulin ELISA kit. Results1. Compared the fingerprints of FTZ preparation, FTZ-serum and rat serum, we concluded that FTZ-serum contained a certain concentration of drug ingredients.2. Compared with control group, the cells of model group contained higher levels of ROS and MDA. Furthermore, the protein expression of Mn-SOD and CAT decreased dramatically. However, FTZ-serum decreased the levels of ROS and MDA, and increased the expression of Mn-SOD and CAT.3. Compared with control group, there was not much change on the anti-apoptotic protein expression of Bcl-2/Bcl-xl in the cells of model group, but the pro-apoptotic protein expression of Bax increased dramatically. Additionally, the activity of caspase 9 and caspase 3 increased in the cells of model group. The rate apotosis was also increased in the model group. However, the rate of apotosis decreased in FTZ-serum groups with the increasing expression of Bcl-2/Bcl-xl and decreasing expression of Bax. The activity of caspase 9 and caspase 3 also decreased.4. Compared with control group, the expression levels of PDX-1 and Glut 2 decreased in the model group, and the glucose stimulated insulin secretion also decreased. However, FTZ-serum could improve the glucose stimulated insulin secretion and increase the expression levels of PDX-1 and Glut 2.Conclusion1. FTZ-serum could improve the oxidative stress in INS-1 cells induced by high glucose through decreasing the levels of ROS and MDA, and increasing the expression of Mn-SOD and CAT.2. FTZ-serum could improve the apoptosis induced by the accumulation of ROS in INS-1cells through decreasing the activity of caspase 9 and caspase 3 which had resulted from increasing expression of anti-apoptotic protein Bcl-2/Bcl-xl and decreasing expression of pro-apoptotic protein Bax.3. FTZ-serum could improve the glucose stimulated insulin secretion through increasing the expression of PDX-1 and Glut 2 proteins.