Study On The Quality Control Of Mitochondrial Protein In Liver Cells Of Rats With Spleen Deficiency Syndrome

Purpose:Traditional Chinese medicine theory believes that spleen qi deficiency can lead to the disturbance of the five viscera including the liver,and points out that spleen deficiency is easily caused by liver,which indicates that the liver can tolerate the interference of spleen qi deficiency without changing its property of inhibiting the spleen,and its modern medical mechanism is unknown.Mitochondria are the main places for aerobic respiration and energy generation of the body cells,which provide energy for various functional activities of the liver.This study focused on the structure and function of hepatocyte mitochondria and their protein quality control to preliminary explore the possible mechanism of liver tolerance to spleen qideficiency.Material and method:1.Experimental animals Sixteen SPF SD male rats,each weighing 190?210 grams,were divided into two groups according to random number table method at the age of 7 weeks.They were divided into two groups:normal group(control group)(8 rats)and spleen qideficiency syndrome group(model group)(8 rats).The experiment was carried out after adaptive feeding for one week2.Model replication and evaluation of spleen qi deficiency syndrome2.1 Model replication Within 15 days of model replication,rats in the experimental group were subjected to 5 cycles of abnormal hunger stimulation(i.e.,1 day of satiation+2 days of fasting without water)and were swimming in water at a temperature of 30?35?every day until they were exhausted.2.2 Model evaluation When the rats in the model group showed emaciation,lack of food,fatigue,fatigue and haggard skin,it was considered that the model was successful.3.Draw materials The rats to be dissected were injected with 10%chloral hydrateintraperitoneally according to the body weight of 0.18 m L per 50g body weight for more than12h on an empty stomach,and the rats were anesthetized.After the abdomen was opened,blood was collected from the abdominal aorta,and the rats were killed by decapitating their heads.The liver tissue was taken from the ice and fixed in 2.5%glutaraldehyde.The rest of the liver tissue was stored in a device at-80?for freezer use.4.Morphological observation of hepatocyte mitochondria 1mm3 of liver tissue fixed in2.5%glutaraldehyde was rinsed with PBS,fixed with 1%osmium acid,dehydrated with ethanol and acetone gradient,embedding polymerization with epoxy resin,sected anddouble-stained with 3%uranium acetate and lead citrate.The morphology of mitochondria in the treated liver was observed by transmission electron microscopy and photographed.5.Liver function test5.1 Determination of glutamic-oxalacetic transaminase(GOT)activity in serum Under the catalysis of GOT,?-ketoglutaric acid and aspartic acid can be converted to glutamic acid and oxaloacetic acid,which are further decarboxylated to pyruvate.Pyruvate can react with 2,4-dinitrophenylhydrazine to produce 2,4-dinitrophenylhydrazine,which appears brownish red under alkaline conditions.The activity of GOT can be calculated according to the change of absorbance at 505nm.5.2 Serum glutamic-pyruvic transaminase(GPT)activity detection GPT can catalyze the amino conversion of alanine and?-ketoglutaric acid to form pyruvate and glutamic acid.2,4-dinitrophenylhydrazine is added to terminate the reaction,and is added to the carbonyl group of ketonic acid to form phenylhydrazone pyruvate;It is reddish brown in alkalinecondition,and can read the absorbance value and calculate the enzyme activity at 505nm.5.3 Detection of superoxide dismutase(SOD)activity in liver tissue SOD can catalyze the disproportional reaction of superoxide anions,and then generate H₂O₂ and O₂.It produces superoxide anions(O^2-)through the reaction system of xanthine and xanthine oxidase,andcan reduce nitrogen-blue tetrazolium to produce blue dirty,the latter can be detected at 560nm.SOD can remove the superoxide anion,thus inhibiting the formation of dirty;The darker the blue of the reaction solution is,the lower the SOD activity is,whereas the higher the activity is.5.4 Detection of malondialdehyde(MDA)activity in liver tissue When oxygen free radicals act on the unsaturated fatty acids of lipids,they can generate lipid peroxide.The lattergradually breaks down into a complex set of compounds,including malondialdehyde.The level of lipid oxidation can be measured by detecting MDA levels.6.The levels of mitochondrial adenosine triphosphate(ATP)and membrane potential(MMP)in hepatocytes were determined The liver mitochondria were extracted and the liver tissue homogenate was prepared according to the instructions of the kit.The supernatant aftertreatment was taken for experiment.The content of ATP in liver mitochondria was detected by colorimetric method,and the changes of membrane potential of liver mitochondria were detected by JC-1 detection method.7.Expression of mitochondrial protein quality control(MPQC)related proteins in hepatocytes Total protein was extracted from liver tissue frozen in-80.Western blot(WB)was used todetect hot shock protein 70(HSP70),Lon protease(Lon protease,Lon P)and caseinolytic protease(CLP)XP complex(including CLPX and CLPP).8.Data processing SPSS17.0 statistical software was used.Data were expressed as mean±standard deviation,and t test was used.P<0.05 was considered to be statistically significant.Results:1.Model replication and evaluation Compared with the control group,the body weight,water intake and food intake,forelimb grip,movement distance and standing times of the rats with spleen qi deficiency syndrome were significantly reduced,and the coat of the rats in the model group was hapless and easy to fall off compared with the normal group.Therefore,model replication is considered successful.2.Morphological changes of hepatocyte mitochondria Mitochondria in the liver cells of the normal group and the model group were distributed near the nucleus in large numbers,with similar structural morphology,oval shape,clear edge and dense interior,without obviousabnormal changes.3.Evaluation of liver function injury The activity of GOT in serum of normal group andmodel group was 3.33±0.46 U/ml,4.09±0.68 U/ml(n=8,P<0.05);The serum GPT activity was 0.86±0.63 U/ml,1.95±0.46 U/ml(n=8,P<0.05);SOD values in liver tissue of normal group and model group were 24728.02±3746.94 U/g,11546.58±1460.72 U/g(n=8,P<0.01);MDA values in liver tissue were 33.53±4.24 nmol/g,40.04±1.76 nmol/g(n=8,P<0.05).4.Changes of mitochondrial ATP and MMP levels in hepatocytes The ATP content in liver tissue of normal group and model group was 1.290±0.186 pmol/mg,0.698±0.132pmol/mg(n=8,P<0.01);The mitochondrial membrane potentials of hepatocytes in the two groups were 1.158±0.195 A.U.,0.526±0.141 A.U.(n=8,P<0.01).5.Expression of MPQC related proteins in liver cells The expression of HSP70 in normal group and model group was 0.599±0.048 and 0.966±0.035,respectively(n=8,P<0.01);Lon P expression was 1.020±0.087 and 0.730±0.080(n=8,P<0.01);CLPX expression was0.836±0.090 and 1.147±0.113(n=8,P<0.01);CLPP expression was 0.940±0.024 and1.288±0.120(n=8,P<0.01).Conclusion:1.In the case of spleen qi deficiency,mitochondrial function of liver cells was damaged to a certain extent,but at the same time,the MPQC effect of liver cells was enhanced to counter the interference caused by spleen qi deficiency.Therefore,this study believed that themechanism of liver tolerance to spleen qi deficiency was related to the enhancement of liver cell MPQC.