Study On The Mechanism Of Plasma Phospholipid And Apoptosis Signal Pathway In Dogs With Multiple Organ Dysfunction Syndrome Treated By Blood Purification

Objective:To observe the effects of CVVHDF on the vital signs,peripheral blood mononuclear cell function and organ function of MODS dogs and their effects on organ pathological lesions;Reveal the molecular mechanism of abnormal expression of apoptosis signal transduction related to endoplasmic reticulum stress response in the course of MODS lesions,and the mechanism of CVVHDF in alleviating apoptosis;aims to find out the differences of plasma biomarkers during the course of MODS and CVVHDF treatment by plasma phospholipid metabolism analysis,and further investigate the effect of CVVHDF on plasma phospholipid metabolism during the treatment of MODS dogs,and provide new ideas for the diagnosis and treatment of MODS.Methods:（1）The Beagle dog was selected as the research object,and the MODS canine model was established through the"second strike"method;They were randomly divided into CVVHDF group and MODS group.The two groups were treated with CVVHDF for 24hours and no treatment as experimental control.T₁ time point is set when the experiment starts,and T₂ time point is set when the MODS dog model is created successfully,and T₃,T₄,and T₅ are set at 6h,12h,and 24h after CVVHDF treatment,respectively.The two experimental animals are monitored the Changes in vital signs（body temperature,blood pressure,heart rate）,organ function（liver function,lung function,and renal function）,peripheral blood mononuclear cell function（antigen presentation,secretory function,and apoptosis）at T1 to T5,respectively.（2）Two groups of experimental dogs were sacrificed at time T5.Liver,lung and kidney tissue samples were taken.HE staining and TUNEL method were used to observe the histopathological changes and histological cell apoptosis differences in various organs.（3）In vivo:Selected liver,lung,and kidney tissue samples from MODS and CVVHDF groups.Quantitative real-time PCR（qRT-PCR）was used to detect the mRNA expression levels of JNK,CHOP,and Caspase-12 in these tissues.（4）In vitro:Four different interfering sequences were designed in vitro for JNK,CHOP and Caspase-12 to complete construction of Human JNK,Human CHOP and Human Caspase-12 shRNA lentivirus vectors;The 293T cells were co-transfected with the constructed lentiviral expression vector and packaging plasmid to complete the virus packaging.The virus stock solution was collected and the titer was measured.Human Umbilical Vein Endothelial Cells（HUVEC）were cultured in vitro and infected with a negative control of lentivirus and a packaged lentivirus.After infection,Western Blot and qRT-PCR methods were used to evaluate the interference effect of the interference vector on the target gene and stable strains were obtained.After being starved,the endoplasmic reticulum stress model was established.The serum of T₁ to T₅ in two groups were used to culture and induced endoplasmic reticulum stress in apoptotic models,Western Blot and qRT-PCR were used to detect mRNA and protein expression of the target gene,apoptotic levels of HUVEC were measured by Flow cytometry.（5）UPLC/Q-TOF MS was established a stable separation condition for plasma phospholipid reversed phase chromatography and mass spectrometry using ESI+and ESI-ion modes.Principal component analysis（PCA）and partial least squares discriminant analysis（PLS-DA）were used to process and analyze the data.Qualitative analysis of the phospholipids in plasma of MODS group and CVVHDF group,aims to find the effect of plasma phospholipid metabolism during the MODS and CVVHDF treatment process.Result:（1）All MODS model dogs showed different degrees of elevated body temperature,accelerated heart rate,and decreased blood pressure.The above indicators of MODS group from T₃ to T₅ time points had significant differences compared with T₁ time point（P<0.05）,those indicators of CVVHDF group improved with the treatment time to varying degrees,and they were all improved at T3^T5 time points compared with MODS group（P<0.05）.All MODS model dogs had different degrees of damage in heart function,liver function,lung function and renal function;The dogs in the CVVHDF group had a better degree of improvement compared to the model（T₂）after treatment with CVVHDF,and there was a significant difference between the T₃ and T₅ time points compared with the MODS group,respectively（P<0.05）.The antigen presentation and secretory function of peripheral blood mononuclear cells in the MODS group were significantly impaired,and the early and late apoptotic levels were significantly increased.Compared with the MODS group,the antigen presentation and secretory function of monocytes in the CVVHDF group was significantly improved,and the early and late apoptotic levels were significantly decreased,and there was a significant difference between the T3 and T5 time points,respectively（P<0.05）.（2）At the tissue level,there were obvious pathological lesions in the liver,lungs and kidneys in the MODS group.The degree of damage in the CVVHDF group was significantly less than that in the MODS group,the apoptotic index of liver,lung and kidney cells in CVVHDF dogs was significantly reduced compared with the MODS group.（3）In vivo:The expression of JNK,CHOP and Caspase-12 mRNA in liver,lung and kidney of two groups of dogs was detected by qRT--PCR.The results showed that the above three genes were expressed in liver,lung and kidney tissues in CVVHDF group.The mRNA expression level was generally lower than MODS group,the difference was statistically significant（P<0.05）.（4）In vitro experiments:Four shRNA interference vectors constructed from JNK,CHOP,and Caspase-12 were sequenced.After alignment,the insert sequences in the recombinant clones were identical to the designed oligo sequences.JNK-shRNA,CHOP-shRNA,Successful construction of Caspase-12-shRNA lentiviral vector.The lentiviral titers of four shRNA lentiviral vectors constructed by JNK,CHOP and Caspase-12 were tested.The results showed that the titer of all constructed lentiviral vectors was higher than 10⁸,which indicated that the lentiviral titers were of good quality.The JNK-shRNA,CHOP-shRNA,and Caspase-12-RNA were transfected into HUVEC,respectively,and validation results showed that all the lentiviruses successfully infected HUVEC.The silencing effect of Western blot and qRT-PCR revealed that the shRNAs JNK-shRNA4,CHOP-shRNA1 and Caspase-12-shRNA1had the best silencing effect in all transfected shRNAs.Therefore,the target cells infected with the above three shRNAs were selected,then a stable strain was constructed successfully.Western Blot and qRT-PCR methods detected that the mRNA,protein,and HUVEC apoptosis levels of JNK,CHOP,and Caspase-12 in CVVHDF group decreased with the prolongation of model time before shRNA interference.The comparison between groups showed that the above-mentioned indicators in the CVVHDF group were significantly lower than those in the MODS group,and the differences were significant at T₂^T₅ time points respectively,suggesting that CVVHDF treatment can reduce the apoptotic effect caused by the"second strike",and the mechanism may be related to reducing the expression of JNK,CHOP and Caspase-12 genes in vivo.What’s more,after shRNA lentiviral specific silencing of JNK,CHOP and Caspase-12 genes,the mRNA,protein and HUVEC apoptosis levels of MODS group and CVVHDF group were significantly lower than those before the interference.The differences in time points were statistically significant,suggesting that CVVHDF treatment can reduce HUVEC apoptosis by inhibiting the expression of JNK,CHOP and Caspase-12.（5）After UPLC/Q-TOF MS detection and data processing by PCA and PLS-DA,it was found that after the establishment of the MODS model（T₂）,lysophosphatidyl choline（LPCs）,ornithine,proline,methionine were showed a downward trend.The levels of carnitine,free fatty acids（FFA）,phosphatidylcholine（PC）,LPEs and bile acids wers shown increased.The levels of orthophosphoric acid,phenylalanine,threonine,tyrosine,carnitine,leucine,and LPE were increased in the MODS group when compare T₄ with T₂,and the levels of acid,citric acid,free fatty acids,and myelin sheaths（SMs）decreased respectively.In the CVVHDF group,the levels of methionine,proline,arginine,and lysine rebounded in T₄compared with T₂ time points,while the levels of carnitine,SMs,and orthophosphoric acid decreased.In addition,,the levels of metabolites such as citric acid,choline,LPCs,LPEs,MAGs,hippuric acid,and p-cresol sulfate were higher in the CVVHDF group than in the MODS group at the T4 time point,while the level of carnitine and PC38:4 had dropped significantly.Conclusion:CVVHDF treatment can significantly improve the disorder of vital signs of MODS dogs,correct major organ functions and histopathological damage,and at the same time improve peripheral blood mononuclear cell antigen presentation,secretion and reduce apoptosis.After treatment with CVVHDF,the expression of JNK,CHOP and Caspase-12 genes and their proteins caused by endoplasmic reticulum stress can be reduced,and the apoptosis of tissue cells can be weakened and the damage of MODS can be inhibited.The abnormal distribution of plasma phospholipid metabolism occurs in the state of MODS.Treatment with CVVHDF can improve the disorder of this phospholipid metabolism and maintain the metabolic balance of the body by regulating a variety of lipid metabolism and amino acid metabolism.