| Object To analyze the effects of carnitine supplementation on serum carnitine levels,fatty acid metabolism levels,liver cell and mitochondrial damage levels in severely scalded rats,and to analyze the protective mechanism of carnitine on liver cell mitochondrial damage by regulating key enzyme activities of lipid metabolism through cell experiments.This provides a theoretical basis for the protective mechanism of mitigating mitochondrial damage in liver cells of patients with severe burns by supplementing exogenous carnitine.Methods1.Animal experiments: By establishing a SD rat model with severe burns and resuscitation with tail vein fluid resuscitation,the changes of serum carnitine,LDH,ALT,AST,OCT,and ?-HB levels in the three groups of rats(control,burn,carnitine)at four time points at 12 h,24h,48 h,and 72 h after the injury were observed as an initial stage experiment.One of the typical time points(24h after injury)was selected for further research,the TG levels in serum and liver tissue were measured,and the changes in TG distribution in liver cells and morphological and pathological changes in liver tissue were observed.The changes of mitochondrial CPT1 enzyme activity level,CPT1?RNA expression level,mitochondrial membrane potential level were detected,and the changes of hepatocyte apoptosis level were detected.Pathological changes of subcellular structure of liver tissue were observed by electron microscope.Three groups of rat liver tissues were taken for high-throughput sequencing of whole RNA omics,and the RNA sequences(Acacb,Acsm5,Fabp4,Pnpla3)that have effects on fat metabolism were selected from them and verified in cell experiments.2.Cell experiments: HepG2 cells were selected as in vitro test cell line.200 ? M etomoxir was used to simulate the inhibition of mitochondrial CPT1 enzyme activity.The changes of carnitine,LDH,ALT,AST,OCT,?-HB in the cell culture supernatant and intracellular TG levels of three groups(control,etomoxir,carnitine)were detected.Changes of TG distribution in cells were observed.Changes of mitochondrial CPT1 enzyme activity levels,CPT1?RNA expression levels were detected.ATP production amount in cells,mitochondrial membrane potential levels and changes in apoptosis levels were detected.The changes in the expression levels of RNA(Acacb,Acsm5,Fabp4,Pnpla3)of genes related to lipid metabolism were observed.3.Clinical observation: The changes of serum carnitine,LDH,ALT,AST,OCT,?-HB,TG levels in patients with severe burns during clinical treatment were observed.Results1.Animal experiments(1)The serum carnitine level of the rats in the burn group decreased significantly at 12 hours after injury,and then showed a slow recovery trend,which was still lower than that of the control group at 72 hours after injury.After carnitine supplementation via intravenous route,the serum carnitine of carnitine group recovered fast than that of the burn group.At 72 hours after injury,the serum carnitine level of carnitine group was close to the control group.At 12 hours after injury,the serum LDH,ALT,and AST levels in the burn group rats reached abnormal peaks,and then gradually decreased,and were still higher than those of the control group at 72 hours after injury.Serum LDH,ALT,and AST levels in the carnitine group also reached abnormal peaks at 12 hours after injury,but their recovery period was shorter than the burn group.At 48 hours after injury,the serum LDH,ALT,and AST levels in the carnitine group were close to those in the control group.At 12 hours after the injury,the serum OCT level of the burn group rats reached an abnormal peak,and then gradually decreased,and was still higher than that in the control group at 72 hours after the injury.The serum OCT level of rats in carnitine group also reached an abnormal peak at 12 hours after injury,and was still higher than that of control group at 72 hours after injury,but the OCT level in burn+carnitnine group at each time point after injury was lower than that of burn group.At 12 hours after the injury,the serum ?-HB level of the rats in the burn group reached an abnormally low point,and then gradually recovered,and was still lower than that in the control group at 72 hours after the injury.The serum ?-HB level in the carnitine group also reached an abnormally low point at 12 hours after injury,and was still lower than that in the control group at 72 hours after injury.(2)There was no significant difference in serum TG levels between the three groups of rats 24 hours after scald.The TG levels in the liver tissue homogenate of the three groups were generally different.The TG levels in the liver tissues of the burn and carnitine groups were higher than those of the control group,and the carnitine group was lower than that of the burn group.The liver cells in the burn group were disordered in arrangement of liver cells,the central vein atrophied,and the manifold area dilated.It can be observed that the infiltration of acute and chronic inflammatory cells were increased in burn group.Small blood vessels are congested.A large number of lipid droplets were seen in the liver tissue of the burn group rats.The pathological changes in the carnitine group were reduced compared with the burn group.The area of oil red staining in burn and carnitine group was more than that of control group,and the area of oil red staining in carnitine group was lower than that of burn group.The mitochondrial CPT1 enzyme activity level of rat liver cells in burn and carnitine groups was lower than that in control group.The carnitine group was higher than the burn group.The level of mitochondrial CPT1?RNA expression in rat liver cells in burn and carnitine groups was higher than that in control group.Carnitine group was higher than burn group.The levels of mitochondrial membrane potentials in burn and carnitine groups were lower than those in control group.Carnitine group was higher than burn group.Under electron microscope,liver tissue of burn group rats showed mitochondrial atrophy,mitochondrial ridges were unclear,and endoplasmic reticulum swelling.The pathological changes of mitochondria and endoplasmic reticulum in liver tissue of rats in carnitine group were less than those in burn group.The number of hepatocyte apoptosis in burn group was higher than that in control group.The carnitine group was similar to the control group.Carnitine group was lower than burn group.(3)The m RNA expression of liver cells in the three groups was statistically different among the three genes Acacb,Fabp4,and Pnpla3.The expression of Fabp4 gene in burn group was higher than that in control group.The expression of Acacb and Pnpla3 genes in carnitine group was higher than that in burn group,while the expression of Fabp4 gene was lower than that in burn group.2.Cell experiments(1)The overall comparison of carnitine levels in the supernatants of the three groups was statistically significant.Carnitine was higher in carnitine group than in control and etomoxir groups.The mitochondrial CPT1 enzyme activity level of etomoxir group cells decreased,and the CPT1 enzyme activity level of carnitine group was similar to that of control group.The levels of CPT1? RNA expression in etomoxir group and carnitine group increased.(2)?-HB levels in the etomoxir group were lower than those in the control group,while ?-HB levels in the carnitine group recovered somewhat,which was close to that in the control group.There was no statistical significance in the overall comparison of the ATP levels of the three groups.The ATP levels in the carnitine group were closer to the control group.(3)The TG level in cells of etomoxir group was higher than that of control group,and the TG level of carnitine group was lower than that of etomoxir group.Through oil red staining,a large number of lipid droplets were formed in the etomoxir group,and a small number of lipid droplets were found in the carnitine group.The area of oil red staining in the etomoxir and carnitine groups was more than that in the control,and the area of oil red staining in the carnitine group was lower than that in the etomoxir group.(4)The level of mitochondrial membrane potential in cells of carnitine group was higher than that of etomoxir group,and the level of OCT in the supernatant of carnitine group was lower than that of etomoxir group.Carnitine group had a lower level of apoptosis than etomoxir group.The levels of LDH,ALT and AST in the supernatant of carnitine group were lower than those in etomoxir group.(5)There were statistically significant differences in RNA expression between the three groups of cells in Acacb and Fabp4 genes.The expression of Acacb gene in carnitine group was higher than that in control and etomoxir groups.The expression of Fabp4 gene in etomoxir and carnitine groups was higher than that in control group,while the expression of Fabp4 gene in carnitine group was lower than that in etomoxir group.3.Clinical Observation At 24 hours after injury,the levels of LDH,ALT,AST,and OCT in serum of patients with severe burns were abnormally increased.Carnitine and ?-HB levels were decreased,and there was no significant difference in TG levels.Conclusion1.Through animal model experiments and clinical observations,we found that carnitine supplementation can quickly and effectively restore serum carnitine levels and restore mitochondrial ketogenic capacity.Carnitine supplementation could restore CPT1 enzyme activity in hepatocytes,promote the increase of CPT1 ? RNA expression,improve fatty acid metabolism,reduce lipid accumulation,restore mitochondrial membrane potential levels,reduce mitochondrial damage,and reduce apoptosis.2.Through the Hep G2 cell model,we found that carnitine may improve the transport of long-chain fatty acids and mitochondrial fatty acid oxidation in liver cells by increasing CPT1 enzyme activity and increasing CPT1?RNA expression.Thus,on the one hand,lipid accumulation in the cytoplasm was reduced,and the formation and accumulation of lipotoxic substances was reduced.On the other hand,the mitochondrial ketogenic ability was improved,and the mitochondrial ATP production was maintained,thereby the stability of mitochondrial membrane potential maintained and mitochondrial damage reduced.Finally,the effect of reducing apoptosis was achieved.3.There was a change in the expression of genes related to fatty acid metabolism in liver tissues after severe burns.The supplementation of carnitine had an effect on the expression of genes related to lipid metabolism.In animal models,it appeared that carnitine had an effect on the m RNA expression of three genes(Acacb,Fabp4,Pnpla3)in liver tissue.In the cell model,it was shown that carnitine had an effect on the expression of Acacb and Fabp4. |
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