| Objective: Non-alcoholic fatty liver disease(NAFLD)is a metabolic disease that currently afflicts a large proportion of the world’s population,and weight loss surgery may alleviate NAFLD to some extent,although the exact mechanism is unknown.Mitochondria play an important role in the development of disease.When abnormal changes in mitochondrial energy metabolism occur,the lipid droplets produced by the liver cannot be transported away,resulting in the accumulation of fat in the liver.In this study,clinical samples were used to measure the corresponding indicators in obese patients.In addition,a mouse weight loss model and a corresponding cellular model were constructed to investigate the effect of weight loss metabolic surgery on mitochondrial function and to discover the specific role of mitochondria in the development of NAFLD using various experimental techniques.Methods: The first part of the study is related to the use of patient specimens.First of all,HE staining was performed on the liver tissue of the patients,and they were divided into two groups according to the results,i.e.,obesity without liver tissue degeneration group(OB CON)and obesity NASH group(OB NASH).Liver tissues from each group were subjected to protein immunoblotting as well as polymerase chain reaction assay techniques to detect the protein expression levels of the corresponding indicator-death-related protein kinase 2(DRAK2)-and the transcript levels at the corresponding m RNA level.The second part of the study was carried out using mouse models.Mice were fed with high-fat chow and high-fat models were constructed for the normal,high-fat,sham-operated and gastric sleeve surgery groups.The mice in the normal group were fed a normal diet,while the remaining three groups were fed a high-fat diet and continued to be fed a high-fat diet for8 weeks after successful membrane creation.The liver tissues and serum of each group of mice were stained with HE and Oil Red O.The TC and TG in the liver tissues and serum were also measured to see if the model was successfully constructed.The protein expression level of DRAK2 and the corresponding m RNA transcript levels were measured by protein immunoblotting and polymerase chain reaction.The third part of the experiment was a cellular experiment in which palmitic acid(PA)was first used to build membranes on L02 and Hep G2 cell lines to construct a high-fat model and to observe whether PA was successful in building membranes by oil red O staining.After successful membrane building,the changes in DRAK2 were detected at the protein level and RNA level respectively.The next step was to design small interfering RNA sequences for PKD and transfect L02 as well as Hep G2 cell lines to test the corresponding transfection efficiency respectively.Once it was determined that Si-PKD was the active chain,transfection was re-performed as well as PA membrane building and divided into normal,PA and transfected groups.The three groups were assayed for DRAK2 and the phase protein expression levels and corresponding m RNA transcript levels were measured by protein immunoblotting and polymerase chain reaction,respectively.In addition to this,mitochondrial membrane potential was measured in the three groups mentioned above,normal,PA and transfected groups,as a reflection of mitochondrial function.In addition,fluorescent probes were used to detect ROS levels in the normal,PA and transfected groups as a reflection of the specific relationship between ROS and DRAK2 and mitochondrial function.In addition,flow cytometry was used to detect apoptosis in the normal,PA and transfected groups,and in this way to illustrate the relationship between DRAK2 and apoptosis.Results: In Part I,significant balloon-like changes were observed in HE sections of patients in the OB NASH group compared with those in the OB CON group.In terms of detection of related indicator DRAK2,the expression level of patients in the OB NASH group was higher than that in the OB CON group at both protein and transcription levels.In the second part,in the HE staining of the mice,we found that there was not much ballooning of the liver tissue in the NC group sections,which were normal liver sections.In the sections of the HFD and SHAM groups,ballooning of the liver was evident and there was some degree of fibrotic changes.Sections in the SG group showed less ballooning than in the HFD and SHAM groups,and the degree of hepatic steatosis was mitigated.In the oil red O staining of mouse liver tissue,there were almost no red lipid droplets in the NC group,while the HFD and SHAM groups had a certain number of red lipid droplets all over the section;the SG group also had red lipid droplets,but they were reduced compared to the HFD and SHAM groups.The expression of DRAK2 was increased in the HFD and SHAM groups compared to the NC group,and decreased to a certain extent after the SG surgery.In the assay of relevant indicator RNAs in mouse liver tissue,the levels of DRAK2 transcripts were higher in the HFD and SHAM groups compared to the NC group,and were decreased in the SG group.We measured the levels of TC and TG in mouse liver tissue and serum respectively.Firstly,in the liver tissue,the TC and TG contents in the NC group were lower than those in the HFD and SHAM groups,and similarly,the TC and TG contents in the SG group were reduced compared to those in the HFD and SHAM groups.Secondly,in the serum we also found higher levels of TC and TG in the serum of the HFD and SHAM groups compared to the NC group,with a decrease in the SG group.In the third part,we constructed high-fat models using PA in L02 and Hep G2 cell lines,and the expression of DRAK2 was higher in the PA group than in the NC group,both at the protein level and at the transcriptional level.In the third part of the experiment,we used small interfering RNA to transfect L02 and Hep G2 cells respectively and successfully knocked down PKD,with both transfection efficiencies above 50%.We used small interfering RNA to transfect L02 and Hep G2 cells respectively to knock down PKD and to assay the protein and transcript levels of the target gene DRAK2.The expression of DRAK2 increased in the PA group and decreased to some extent in the transfected group after giving transfection to both groups of cells.In the apoptosis assay,the PA group had the highest number of apoptosis among the three subgroups,and the apoptosis rate decreased after the administration of transfection,but was still higher than that of the NC group.During the detection of mitochondrial membrane potential,there are two kinds of fluorescence,red and green,in the same picture.The higher the percentage of green fluorescence,the lower the mitochondrial membrane potential,and the lower the percentage of green fluorescence,the higher the mitochondrial membrane potential.We did qualitative analysis and to the naked eye,the percentage of green fluorescence was higher in the PA group than in the NC group and decreased in the transfected group;i.e.the membrane potential was lower in the PA group compared to the NC group and increased after transfection.We also used a fluorescent probe to detect the ROS content.The red fluorescence,which is the ROS bound by the probe,was much greater in the PA group than in the NC and transfected groups,i.e.the ROS content increased after the cells were treated with PA and decreased in the transfected group.Conclusion: In patients with non-alcoholic fatty liver disease,the higher the degree of steatosis,the higher the expression of DRAK2.This is mainly because,during the development of non-alcoholic fatty liver disease,certain changes such as oxidative stress and inflammation occur in the body,leading to the production of large amounts of ROS in the body,of which ROS,through stimulation of PKD,leads to increased expression of DRAK2,which in turn affects mitochondrial function.When the energy produced by mitochondria is reduced,lipid droplets in the liver cannot be transported out,thus aggravating NAFLD.However,weight loss surgery can reduce the amount of ROS released by lowering the level of oxidative stress in the body,improving mitochondrial function and alleviating the degree of liver steatosis. |
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