Protective Effect And Mechanism Of Octreotide On Mouse Islet Cells Min6 In Early Hypoxic Environment

Objective: To isolate and purify pancreatic endocrine cells from mice.To establish a C57 BL /6 diabetic mouse model and a diabetic mouse subrenal islet transplantation model,then use octreotide as an intervention factor to observe the effects of early drugs on blood glucose and glucose tolerance after transplantation,and to investigate the protective effect of octreotide on islet cells in mice in early hypoxic environment and its mechanisms.Methods: In vivo experiment :Healthy adult male SPF C57 BL /6 mice were taken,and the pancreatic cells of the mice were digested and separated retrograde cholangiopancreatic duct perfusion with collagenase V after anesthesia.Then we isolated them with Histopaque-10771 lymphocyte isolation solution.Dithizone(DTZ)was used to specifically stain mice islet cells and count and analyze the purity of pancreatic endocrine cells.Cells were stained by trypan blue to calculate the activity of pancreatic endocrine cells.30 healthy adult(8-12 weeks old)male SPF C57 BL /6 mice were selected,and we use 1% streptozotocin STZ citric acid solution to inject them intraperitoneally.The C57 BL /6 mice were injected at 170 mg /kg and induced into diabetic mice.Successfully induced diabetic C57 BL /6 mice were selected(n = 26).The donor pancreas was digested,separated and purified using the above method,and the upper pole under the left renal capsule of diabetic mice was implanted at a quantity of 200 per mouse.Then we processe the capsule,and then suture the incision.All mice were randomly divided into two groups:the simple transplantation group(control group,n = 13)and the transplantation plus medicine group(experimental group,n = 13).The mice in the experimental group were subcutaneously injected with 50 μg /Kg of octreotide three times a day;the mice in the control group were injected with the same amount of normal saline three times a day for 14 days.Starting from the first day the sixth day after surgery,blood samples were collected from the tail vein of two groups of mice at a fixed time daily and blood glucose was monitored.From the seventh day to the fourteenth day,blood glucose was detected every2-4 days.It was defined that diabetes was cured if blood glucose was lower than11.1 mmol /L twice in a row.The left kidney of mice was removed on the 14 th day,and the blood glucose was detected every day for 7 days.It was defined as diabetes recurrence if the blood glucose was more than 21 mmol /L twice in a row.On the fifth day after transplantation,a glucose tolerance test was performed on diabetic healers from two groups of mice(control group a = 3and experimental group a = 3),and a 0.3 g /ml glucose solution was injected intraperitoneally at a dose of 1 g /kg.Tail vein blood was collected at 0 min,30 min,60 min,90 min,and 120 min after injection,and the blood glucose concentration of the mice was measured by a blood glucose meter.In vitriol experiment : Min6 cells were randomly divided into four groups:normoxia group,hypoxia group,normoxia + octreotide group and hypoxia + octreotide group.The normoxia + octreotide group and hypoxia + octreotide group were treated with octreotide at a concentration of 10-8 mol/L.The normoxic group and normoxia + octreotide group were placed in a 37 ℃,21% O2,5% CO2 carbon dioxide incubator,and the hypoxia group and hypoxia + octreotide group were placed at a 37℃,1 % O2,5 % CO2,94 % N2 hypoxia incubator for 12 h.Cells and the culture supernatant of all groups were collected respectively.Western blot was used to find out the optimal concentration of octreotide on the expression of HIF-1α in pancreatic endocrine Min6 cells,and the cell proliferation activity after octreotide treatment was detected by the CCK-8 method.HIF-1α was detected by Western Blot Expression,enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of VEGF in pancreatic endocrine cells,Western blot was used to detect the expression of target proteins such as Bcl-2,LC3,and cell apoptosis was detected by flow cytometry.Result: In vivo experiment: At least(140 ± 20)islets were collected from each C57 BL /6 mouse after digestion,isolation and purification.After DTZ staining,a purity greater than 90% was detected.After trypan blue staining,the activity aws greater than 90%.From the 1st to 20 th days after islet transplantation,the blood glucose of the mice in the simple transplantation group were(24.62 ± 0.02)mmol /L、(13.79 ± 0.04)mmol /L、(11.11 ±0.05)mmol /L、(11.18 ± 0.04)mmol /L、(10.88 ± 0.05)mmol /L、(11.06± 0.05)mmol /L、(10.08 ± 0.05)mmol /L、(8.43 ± 0.03)mmol /L、(8.59 ± 0.04)mmol /L、(17.71 ± 0.05)mmol /L、(20.64 ± 0.06)mmol /L、(24.10 ± 0.06)mmol /L,respectively.The blood glucose of mice in the transplantation and dosing group were(24.61 ± 0.06)mmol /L、(9.34± 0.06)mmol /L、(8.08 ± 0.01)mmol /L、(7.41 ± 0.05)mmol /L、(7.35 ± 0.03)mmol /L、(6.99 ± 0.03)mmol /L、(6.73 ± 0.06)mmol/L、(7.07 ±0.03)mmol/L(6.91 ± 0.02)mmol /L、(16.97 ± 0.06)mmol /L、(20.03 ±0.04)mmol /L、(24.26 ± 0.02)mmol /L,respectively.The blood glucose of the two groups of mice began to drop on the second day after the islet transplantation.The blood glucose recovery time of the experimental group was earlier than that of the control group,and the overall blood glucose level of the experimental group was lower than the control group(p < 0.05).After the removal of the left kidney,the blood glucose of the two groups increased rapidly.In the glucose tolerance test,the blood glucose values of the mice in the control group A(n = 3)at 0 min,15 min,30 min,60 min,90 min and 120 min after intraperitoneal glucose injection were(8.05± 0.13)mmol /L,(23.32 ± 0.04)mmol /L,(19.23 ± 0.07)mmol /L,(13.86 ± 0.02)mmol /L,(11.24 ± 0.07)mmol /L,(10.54 ± 0.02)mmol /L,respectively.The blood glucose levels of mice in experiment group A(n =3)were(7.03 ± 0.10)mmol /L,(18.53 ± 0.04)mmol /L,(14.20 ± 0.01)mmol /L,(11.11 ± 0.01)mmol /L,(8.09 ± 0.02)mmol /L,(6.99 ± 0.07)mmol /L,respectively.In the control group A and the experimental group A,after the intraperitoneal injection of glucose,the blood glucose increased rapidly,and then began to decrease after 15 minutes.The overall blood glucose level of the experimental group A was lower than that of the control group A(p< 0.05).In vitriol experiment: Different concentrations of octreotide(10-8 mmol/L,10-9 mmol /L,10-10 mmol /L)were used to treat the mice pancreatic endocrine Min6 cells under normoxia and hypoxia conditions.The expressions of HIF-1α in the normoxia culture group,normoxia 10-8 mmol /L octreotide group,normoxia 10-9 mmol /L octreotide group,normoxic 10-10 mmol /L octreotide group were determined to be(0.27 ± 0.0067),(0.19 ± 0.0068),(0.22 ± 0.0032),(0.23 ± 0.0070),the expressions of HIF-1α hypoxia culture group,hypoxia 10-8 mmol / L octreotide group,hypoxia 10-9 mmol / L octreotide group,hypoxia 10-10 mmol / L octreotide group was(1.05 ±0.0048),(0.38 ± 0.0072),(0.41 ± 0.0068),(0.43 ± 0.0063),The expressions of HIF-1α in cells of hypoxia groups were higher than that in normoxia groups,and the expressions of HIF-1α in cells of each dosing group were lower than that in the same oxygen concentration culture group(p<0.05).In the CCK-8 test,the OD values of the normoxia group after culture for 1 h,3 h,6 h,9 h and 12 h were(0.789 ± 0.034),(0.753 ± 0.001),(0.941 ± 0.028),(1.234 ± 0.012)and(1.280 ± 0.037).The OD values of the normoxia + octreotide group were(0.752 ± 0.025),(0.713 ± 0.008),(0.828 ± 0.012),(1.063 ± 0.030)and(1.145 ± 0.027).The OD values of the hypoxia group were(1.533 ± 0.020),(1.666 ± 0.005),(1.967 ± 0.023),(2.416 ± 0.030),and(2.387 ± 0.016).The OD values of the hypoxia +octreotide group were(1.348 ± 0.023),(1.446 ± 0.014),(1.780 ± 0.010),(2.101 ± 0.084)and(2.010 ± 0.043).Compared with the groups cultured with the same oxygen concentration,the cell proliferation of each medicated group was reduced(p<0.05).The VEGF expressions in the cells of normoxia group,normoxia + octreotide group,hypoxia group,and hypoxia + octreotide group were(77.27 ± 2.2075)pg / ml,(76.75 ± 1.7547)pg / ml,(76.17 ±2.3538)pg / ml,(65.50 ± 1.471)pg / ml,the expression of VEGF in the hypoxia + octreotide group was lower than that in the hypoxia culture group(p<0.05).The apoptotic rates of the cells in the normoxia group,normoxia +octreotide group,hypoxia group,and hypoxia + octreotide group were(10.46± 0.6873),(9.22 ± 0.2768),(14.57 ± 0.3799),(8.69 ± 0.0115),the apoptotic rate of each dosing group was lower than that of the same oxygen concentration culture group(p<0.05).The Bcl-2 expressions in the normoxia group,normoxic + octreotide group,hypoxia group and hypoxia + octreotide group were(0.23 ± 0.0017),(0.25 ± 0.0021),(0.30 ± 0.0029),(0.37 ±0.0021),the relative expressions of LC3 were(1.16 ± 0.0033),(1.22 ±0.0098),(1.26 ± 0.0044),and(1.05 ± 0.0017).The Bcl-2 expression of each medicated group was higher than that in the culture group with the same oxygen concentration,and the expression level of LC3 in the hypoxia +octreotide group was lower than that in the hypoxia group.Conclusion: Our research team has mastered the methods of digestion,isolation and purification of mice pancreatic endocrine cells to obtain islets with higher purity and activity,which will prepare for the next step of in vivo and in vitro drug research.Intraperitoneal injection of 170 mg / kg streptozotocin STZ citric acid solution can successfully induce a C57 BL / 6mice diabetes model.We have mastered the methods of isolation and purification of islets,and implanted the islets under the left kidney capsule of diabetic mice in order to establish an animal model of islet transplantation.Using octreotide in the diabetic mouse transplantation models after islet transplantation can accelerate the time for postoperative blood glucose return to normal,keep blood glucose at a relatively low level,and increase the tolerance to high glucose in postoperative mice.The vitriol experiment may prove that hypoxia environment can promote the expression of HIF-1α in Min6 cells.In normoxia and hypoxia conditions,octreotide can down-regulate the expression of HIF-1α in mice pancreatic endocrine cells Min6,and inhibit the proliferation of Min6 cells to a certain extent.It can also inhibit the expression of VEGF in Min6 cells in hypoxia environment,up-regulate the Bcl-2 expression of Min6 cells in hypoxic environment,reduce its apoptotic rate,and down-regulate the level of LC3,and reduce the level of autophagy.