Methyl Eugenol Improves The Survival And Function Of Islet Cells Injured By Hypoxia/Reoxygenation

Objective: In this study,an in vitro hypoxia/reoxygenation model was established to simulate islet transplantation ischemia-reperfusion injury,and to explore the protective effect of methyl eugenol on islet cell survival and its possible mechanism after hypoxia and reoxygenation injury.Methods: Islets were isolated and purified from 6-8w male BALB/C mice and divided into four groups:(1)Normal control group: no special treatment.(2)Hypoxia/reoxygenation group(H/R): Hypoxia/reoxygenation treatment.(3)DMSO solvent group: treated with DMSO solvent and hypoxia/reoxygenation.(4)Methyl eugenol(Me)administration group:treated with Methyl eugenol and hypoxia/reoxygenation.The viability of islet cells in each group was detected by acridine orange(AO)/propidium iodide(PI)double staining.Finally,the function of islet cells,insulin secretion,was detected by ELISA.To further explore how Methyl eugenol protects islet cells.Min6 cells were divided into four groups:(1)Normal control(Normal control): without any special treatment.(2)Hypoxia/reoxygenation group(H/R): Hypoxia/reoxygenation treatment.(3)DMSO solvent group: DMSO solvent and anoxia/reoxygenation treatment.(4)Methyl eugenol administration group: methyl eugenol and hypoxia/reoxygenation treatment were given.CCK8 was used to detect the effect of methyl eugenol on the viability of Min6 cells in normal culture and hypoxia/reoxygenation treatment under a certain concentration gradient.The apoptosis rate of Min6 cells in each group was measured by flow cytometry(Annexin V-FITC/PI double staining kit).The proportion of apoptotic cells was observed by Hoechst 33342 nuclear staining.Western blot was used to detect the expression of p-JNK,p-p38,JNK,p38,Bcl-2,Bax.Results: Compared with the normal group,the primary islet cell viability of the H/R treatment decreased.After Me treatment,the islet cell viability increased compared with the H/R group and the DMSO solvent group,and the insulin secretion function was improved.After me treatment,the normal Min6 cells had no significant effect on the viability of the cells in a certain concentration range.After H/R treatment,the viability of Min6 cells was increased compared with that of H/R group.Compared with H/R group,the total apoptosis rate of cells in Me group decreased,the expression of p-JNK and p-p38 was inhibited,and the Bcl-2/bax ratio increased.Conclusion: H/R treatment aggravated the damage of primary islet cells and Min6 cells,decreased the viability of islet cells and the function of insulin secretion,and increased the total apoptosis rate of Min6 cells.After treatment with Me,the viability and function of islet cells were improved compared with those of H/R group and DMSO group.In Min6 cell line,after treated with H/R,Me could inhibit the activation of JNK and p38 MAPK signal,change the levels of proteins related to apoptosis in the downstream of JNK and p38 MAPK,increase the ratio of Bcl-2/Bax,and then inhibit the apoptosis of cells.