| BackgroundSevere burns are often complicated by glucose metabolic disorders due to thermal damage,secretion of inflammatory cytokines,elevated levels of stress hormones,and absorption of necrotic substances from the wound.Hyperglycemia is the main form of glucose metabolism disorder,which aggravates immune damage,increases susceptibility to infection,and delays wound repair,closely related to the prognosis of severe burns.Our previous research found that hyperglycemia after severe burns was related to the damage of islet structure and function,as well as the relative deficiency of insulin synthesis and secretion.Hypoxia of tissues and organs caused by shock is the main pathophysiological process in the early stage of severe burns.Islet beta cells are susceptible to hypoxic stress,which results in increased production of reactive oxygen species.While excessive reactive oxygen species can damage the function of pancreaticβcells and induceβcell apoptosis.Therefore,this study intends to explore the occurrence and molecular biological mechanism of the mitochondrial oxidative stress injury in pancreatic islets of mice after severe burns.Methods1.Occurrence of mitochondrial oxidative stress injury in pancreatic islets and its effect on islet function after severe burnsC57BL/6 mice were used to establish a third-degree burn model with a burn area of 30%TBSA.The mice was randomized into a sham group and a burn group.The fasting blood glucose of the mice in each group was detected at 24 hours after injury.Islets were isolated to conduct the glucose-stimulated insulin secretion experiment.The electron density of insulin granules in the isletβcells of the mice in each group was observed by transmission electron microscope,and the dense core and vesicle diameter ratio were calculated.The expression of insulin was detected after islets from two groups incubated with high glucose.The levels of reactive oxygen species in islet cells of mice in the sham group and burn group were detected.For the production of reactive oxygen species generation:the expressions of Nox1,Nox2 and Nox4,the activities of mitochondrial respiratory chain complex I and complex III,mitochondrial superoxide levels and mitochondria structural of isletβ-cell were detected.For the reactive oxygen species detoxication system:the concentration of NADPH,GSH/total glutathione ratio,the expressions of Trx1,Trx R1,Trx2,Trx R2,Txnip,the activity of Trx R2,and the expression and activity Mn-SOD were detected.Sodium pyruvate was used to improve mitochondrial reactive oxygen species levels,mitochondrial superoxide levels,the activity of mitochondrial respiratory chain complex III,the concentration of NADPH,the expressions and activities of Trx R2 and Mn-SOD,mitochondrial structure were detected,the ratio of isletβ-cell dense core and vesicle diameter was calculated,and the fasting blood glucose,insulin secretion ofβ-cell were detected.The survival rate of mice treated with sodium pyruvate was evaluated.2.Mechanism of oxidative stress injury in pancreatic islets after severe burns（1）The mechanism of Sirt3 regulating mitochondrial oxidative stress after severe burnsC57BL/6 mice were used to establish a third-degree burn model with a burn area of 30%TBSA.The concentration of NAD+and the expression of Sirt3 were detected at 24 hours after injury.Islet hypoxia was assessed using hypoxia probes and Hif-1αexpression levels.An in-vitro hypoxia model（O2 1%,CO2 5%,N2 94%）was established using mice pancreatic isletβ-cell,MIN6 cells.The NAD+concentration and Sirt3 expression were detected after hypoxia,and the cell function was evaluated by a glucose-stimulated insulin secretion assay.Then,the effect of hypoxia on mitochondrial function of MIN6 cells and the effect of severe burns on mitochondrial function of mice’islet cells were explored.The mitochondrial structure changes in MIN6 cells after hypoxia and in pancreatic isletβcells of mice post severe burns were detected.The level of mitochondrial superoxide was detected.The expressions,the acetylation levels and the activities of Mn-SOD and Idh2 were detected.Mitochondrial membrane potential and the release of cytochrome c were detected,the expressions of Bax,Bcl2,Cleaved caspase3 and Cleaved caspase 9 were detected,and the cell apoptosis rate was detected.The effects of Sirt3 on mitochondrial function in MIN6 cells and pancreatic islet cells of mice were explored.Sirt3 was knocked down in MIN6 cells by si RNA,and wild-type and Sirt3KO mice were used to establish a third-degree burn model with a burn area of 30%TBSA.The mitochondrial structure,the mitochondrial superoxide level,and the acetylation levels and activities Mn-SOD and Idh2 were detected.The mitochondrial membrane potential,the release of cytochrome c,and the expression of Bax,Bcl2,Cleaved caspase 3 and Cleaved caspase 9,and cell apoptosis rate were detected.The glucose-stimulated insulin secretion experiment was conducted to evaluate the function of MIN6 and isletβcells,and the fasting blood glucose level was detected.A MIN6 cell line overexpressing Sirt3 was established.The mitochondrial structure,the mitochondrial superoxide level,and the acetylation levels and activities of Mn-SOD and Idh2 were detected.The mitochondrial membrane potential,the release of cytochrome c,and the expressions of Bax,Bcl2,Cleaved caspase 3 and Cleaved caspase 9 were detected.The glucose-stimulated insulin secretion assay was conducted to evaluate the cell function.（2）The mechanism of Sirt1 regulating mitochondrial function after severe burnsC57BL/6 mice were used to establish a third-degree burn model with a burn area of 30%TBSA.The mice were randomized into sham group,burn group,burn+NMN group and burn+NMN+EX-527 group.The concentration of NAD+,the expression of Sirt1,the phosphorylation level and acetylation level of NF-κB,the apoptosis rate and the expressions of Bax,Bcl2,Cleaved caspase 3 and Cleaved caspase 9 in the four groups of mice islets were detected at 24 hours post severe burns.The mitochondrial superoxide level,mitochondrial membrane potential,ATP concentration and mitochondrial DNA（mt DNA）content of pancreatic islets in the four groups were detected,and the protein expression levels of Ucp P2,Pgc1-α,Mn-SOD and nuclear respiratory factor 1（Nrf-1）,nuclear respiratory factor 2（Nrf-2）,and the expression levels of mitochondrial transcription factor A（Tfam）and m RNA.The glucose-stimulated insulin secretion ability of isletβcells in four groups of mice were detected,and the fasting blood glucose levels were detected.3.The mechanism of isletβ-cell dysfunction after severe burns at the single-cell levelCre-Lox P recombinase system was used to construct Ins2-Cre mice and Rosa26-LSL-td Tomato mice as breeding mice to breed.The mice expressing the fluorescent protein td Tomato were used to establish a third-degree burn model with a burn area of 30%TBSA.Pancreatic islets of the mice were isolated,isletβcells were obtained by fluorescence-activated cell sorting,and single-cell RNA sequencing was performed.Cell Ranger and R language were used to perform quality statistics,reference genome alignment,cell number statistics,gene expression quantification,low-quality cell filtering,and cell cluster classification.Cell clusters were annotated based on known marker genes for further analysis of the differential genes and their functions in isletβcell subsets were analyzed simultaneously.The differentially expressed genes of pancreatic isletβcells in the sham group and the burn group were analyzed,and the GO enrichment analysis was performed accordingly.C57BL/6 mice were used to prepare a third-degree burn model with a burn area of 30%TBSA.The islets of the two groups of mice were extracted for Bulk-cell RNA sequencing,and GO enrichment analysis of differentially expressed genes was performed.Results1.Occurrence of mitochondrial oxidative stress injury in pancreatic islets and its effect on islet function after severe burnsAfter 24 hours of severe burns,the level of fasting blood glucose of the mice increased.The insulin secretion level stimulated by high glucose of pancreaticβcells decreased,the density of insulin granules in pancreaticβcells decreased,and the ratio of dense core to vesicle diameter was significantly reduced.Insulin synthesis of islet beta cells in mice decreased,and the level of reactive oxygen species in islet cells increased.For the reactive oxygen species production:at 24 hours after severe burns,the expressions of Nox1 and Nox2 in pancreatic islets did not change,and the expression of Nox4 decreased,suggesting that the Nox system was not the main source of reactive oxygen species in islets of mice after severe burns.After severe burns,the level of mitochondrial superoxide was significantly increased,the mitochondrial respiratory chain complex I increased,and the activity of complex III decreased in mouse pancreatic islets,the mitochondrial structure of isletβcells was abnormal,the mitochondrial cristae were shortened,side-shifted and mitochondria were swollen,cavitation,matrix electron density decreased,suggesting that the mitochondrial respiratory chain is the main way of producing reactive oxygen species in pancreatic islets after severe burns.For the reactive oxygen species detoxication system,there was no significant change in islet GSH/total glutathione after severe burns.The concentration of NADPH reduced significantly.The expressions of Trx1 and Trx R1 did not change,while the expressions Trx2 and Trx R2 reduced significantly,and the expression Txnip significantly increased,accompanied by decreased Trx R2 activity.At the same time,the expression and activity of Mn-SOD were significantly decreased after severe burns.After intervention with sodium pyruvate,the level of mitochondrial reactive oxygen species in mouse pancreatic islets reduced,the normal structure of isletβ-cell mitochondria was maintained,the concentration of islet NADPH increased,the activity of Trx R2 increased,and the expression and activity of Mn-SOD were upregulated.At the same time,administration of sodium pyruvate could improve the insulin secretion stimulated by glucose in pancreaticβcells,reduce post-injury fasting blood glucose,and improve the survival rate.2.Mechanism of oxidative stress injury in pancreatic islets after severe burns（1）The mechanism of Sirt3 regulating mitochondrial oxidative stress after severe burnsTwenty-four hours after severe burns,the expression of Sirt3 in islets of mice was significantly reduced,and the concentration of NAD+was significantly reduced.At the same time,the expression of islet Hif-1αincreased.Therefore,we purified the hypoxia single factor and established an in vitro hypoxia model with the MIN6 cell line After 24 hours of hypoxia,the glucose-stimulated islet secretion capacity,Sirt3 expression,and NAD+concentration were significantly reduced.At the same time,hypoxia and severe burns led to the destruction of mitochondrial structure in MIN6 cells and isletβcells,increased mitochondrial superoxide levels The expressions of significantly decreased,the acetylation levels of Mn-SOD and Idh2significantly increased,and the activity significantly decreased.In addition,hypoxia and severe burns resulted in mitochondrial membrane potential decreasing,induced the release of cytochrome c from mitochondria to the cytoplasm,and the expression of Bcl2 significantly decreased,the expressions of Bax,Cleaved caspase 3,and Cleaved caspase 9 significantly increased.The apoptosis rate was also significantly increased.Afterwards,we explored the effect of Sirt3 on mitochondrial function in MIN6 cells and islet cells.Small interfering RNA in vitro was used to knockdown Sirt3 in MIN6 cells.Decreased expression of Sirt3 induced a significant increase in MIN6 mitochondrial superoxide level.The acetylation levels of Mn-SOD and Idh2 increased significantly,and activities of Mn-SOD and Idh2 significantly decreased.The decreased expression of Sirt3 led to a significant decrease in mitochondrial membrane potential in MIN6 cells,and induced an increased release of cytochrome c from mitochondria into the cytoplasm.The expression of Bcl2 was significantly decreased,the expressions of Bax,Cleaved caspase 3,and Cleaved caspase 9 were significantly increased,and the apoptosis rate was significantly increased.The decreased expression of Sirt3 could lead to a significant decrease in the glucose-stimulated insulin secretion capacity of MIN6 cells.In addition,In addition,we used wild-type and Sirt3 KO mice to establish a severe burn model.We observed a phenomenon consistent with the in-vitro experiments in the islet cells of Sirt3 KO mice in the sham group.Burns could lead to further impairment in the islet function of Sirt3 KO mice.Finally,the overexpression of Sirt3 could reverse the damage to the mitochondrial structure caused by hypoxia for 24 hours,significantly reduce mitochondrial superoxide levels,reduce acetylation levels of Mn-SOD and Idh2,and increase the activity of Mn-SOD and Idh2.Overexpression of Sirt3 could also reverse the hypoxia-induced decrease of mitochondrial membrane potential in MIN6 cells,reduce the release of cytochrome c from mitochondria to the cytoplasm after hypoxia treatment,significantly increase the expression level of Bcl2,while reduce the expression levels of Bax,Cleaved caspase 3,and Cleaved caspase 9.At the meantime,overexpression of Sirt3 could improve the glucose-stimulated insulin secretion capacity of MIN6 cells under the situation of hypoxia.The role of Sirt3 in anti-oxidative stress,anti-apoptosis and improving glucose-stimulated insulin secretion ability after hypoxia can be inhibited by Sirt3 inhibitor 3-TYP.（2）The mechanism of Sirt1 regulating mitochondrial function after severe burnsTwenty-four hours after severe burns,the concentration of NAD+in the islets of mice was significantly decreased,the expression of Sirt1 was decreased,the phosphorylation and acetylation of NF-κB p65 were significantly increased,the apoptosis rate of islet cells was significantly increased,the expression of Bcl2 was decreased,and the expressions of Bax,Cleaved caspase 3,Cleaved caspase 9 showed an upward trend.After NMN treatment,the NAD+concentration and Sirt1 expression were significantly increased,the phosphorylation and acetylation levels of NF-κB p65 were significantly decreased,and the apoptosis rate in islet cells decreased,the expression of Bcl2 increased,and the expressions of Bax,Cleaved caspase3,and Cleaved caspase 9 showed a downward trend,the Sirt1 inhibitor EX-527 could inhibit the protective effect of NMN.After severe burns,the level of mitochondrial superoxide in pancreatic islets increased,the mitochondrial membrane potential decreased significantly,the concentration of ATP reduced significantly,and the content of mt DNA decreased significantly.The expressions of Nrf-1,Nrf-2 and Tfam m RNA were also significantly reduced.NMN treatment could reverse the above-mentioned changes in islet cells caused by severe burns,and the protective effect could be inhibited by EX-527.After NMN enhanced Sirt1 activity and increased Sirt1expression,the islet function of mice was improved,the ability of glucose-stimulated insulin secretion was enhanced,and the fasting blood glucose of mice reduced significantly,which could be inhibited by EX-527.3.The mechanism of isletβ-cell dysfunction after severe burns at the single-cell levelUMAP was used to analyze the single-cell RNA sequencing data,and according to the marker genes of various islet cells,they were annotated asβcells,αcells,delta cells,acinar cells,and macrophage quiescent stellate cells.Through further analysis of the marker genes of eachβcell subset,we annotated them as Matureβcell,Immatureβcell,UPR hiβcell,Stressedβcell,Replicatingβcell,RNA splicing activeβcell,Rp hiβcell,Mt hiβcell,and Quiescentβcell,a total of 9 subgroups.The highly expressed genes of the three subgroups of Immatureβcell,UPR hiβcell and Stressedβcell were mainly related to insulin secretion and cell stress.At the same time,a group of cells with the same gene expression characteristics as the Ppy lineageβcells were found in the Immatureβcell subgroup,with low expression of Ucn3 and Glut2,and high expression of Ppy and Tspan8.After severe burns,the proportion of three subgroups of Immatureβcell,UPRβcell and Stressedβcell decreased,and the proportion of Replicatingβcell subgroup increased.In the meantime,the results of bulk-cell RNA-seq suggested that pancreatic islet dysfunction after severe burns might be related to cell apoptosis.Conclusion1.Twenty-four hours after severe burn,the mitochondrial structure and function of isletβ-cells in mice were impaired,and the activity of mitochondrial respiratory chain complex III decreased,resulting in increased mitochondrial reactive oxygen species production.At the same time,mitochondrial thioredoxin system was inhibited,and the expression and activity of Mn-SOD were reduced,leading to a reduced mitochondrial reactive oxygen species clearance and mitochondrial oxidative stress damage in pancreatic islet cells,resulting in reduced insulin synthesis and secretion of isletβcells,involving in hyperglycemia.Sodium pyruvate could play an anti-mitochondrial oxidative stress role after injury,improve islet function after injury,and improve the survival rate of mice.2.Severe burns could lead to decreased expression of Sirt1 and Sirt3 in pancreatic islets,mediating mitochondrial dysfunction including mitochondrial oxidative stress damage,increased mitochondrial apoptosis,decreased ATP synthesis,and decreased mitochondrial biosynthesis through downstream proteins or pathways,which led to the decreased synthesis and secretion of insulin and revolved in the occurrence of hyperglycemia post severe burns.3.Through single-cell RNA sequencing,mouse pancreaticβcells could be divided into 9subgroups:Matureβcells,Immatureβcells,UPR hiβcells,Stressedβcells,RNA splicing-activeβcells,Rp hiβcells,Mt hiβcell,Replicatingβcell,and Quiescentβcell.These threeβcell subsets:UPR hiβcell,Stressedβcell and Immatureβcell,mainly involved to the function of insulin secretion.After severe burns,the proportion of these three groups of cells decreased,and the genes related to cell apoptosis were enriched in pancreatic islets after injury,which may be related to the dysfunction of pancreatic islets after burns. |
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