Based On Proteomics To Explore The Effect Of The Method Of Strengthening The Spleen, Removing Phlegm And Removing Blood Stasis On The Changes Of Intestinal Differential Protein And Energy Metabolism In AS Bama Pigs With Spleen Deficiency And Phlegm

Purpose : Through the analysis of the theoretical core of "spleen dominating the circulation" and the relationship between the functional spleen and the substantial small intestine of TCM and the formation of phlegm,turbidity and blood stasis syndrome,the relationship between the modern biological changes of small intestine and the occurrence and development of AS is clarified.On this basis,we established AS Bama piglet model of spleen deficiency and phlegm turbid syndrome,and used i TRAQ technology to find differential expression protein,especially mitochondrial energy metabolism as the main cut-off point,to further explore the influence and mechanism of spleen strengthening,phlegm removing and blood stasis removing method.Material and method : In the first part of the paper,the relationship between the digestion and absorption of functional spleen and substantial small intestine and the formation of phlegm,turbidity and blood stasis syndrome in the body was explored,and the relationship between spleen and heart,heart and small intestine,spleen and small intestine and the formation of AS were discussed.In the second and third papers,9 SPF healthy half year old male Bama minipigs were randomly divided into normal control group(normal group),spleen deficiency phlegm turbid AS group(model group)and Jianpi Qutan Huayu TCM intervention group(intervention group),with 3 pigs in each group.The normal group was fed basic diet for 48 weeks.The model group and the intervention group adopted the TCM compound factor modeling method,that is,the model of spleen deficiency caused by diet and overwork,combined with the modern medicine coronary artery endothelial injury surgery to create AS model.The model group and the intervention group were fed with high-fat diet,and the intervention group was fed with the patent prescription Jianpi Qutan Huayu in the 25 th week.At the end of 48 weeks,the small intestine tissue was obtained by laparotomy.The morphology of intestinal epithelium was observed under the second microscope.The total protein of small intestine was separated and extracted.After being labeled with i TRAQ reagent,it was detected by mass spectrometry and analyzed by software.The proteomic difference between model group and intervention group was analyzed by LC-ESI-MS / MSmethod.The difference protein was enriched based on go function annotation,enrichment and KEGG pathway,and the significantly enriched protein was screened.In order to further verify the changes of energy metabolism,colorimetry was used to observe the changes of activity of mitochondrial respiratory chain complex Ⅰ,Ⅱ,Ⅲ,Ⅳ,Ⅴ in small intestine of pigs in each group,and the amount of ATP synthetase was measured.Western blot was used to detect the protein expression of IDH,Na +-K +-ATPase and ATP5.Results:In the first part of the paper,the relationship between spleen,heart,small intestine and AS is demonstrated,which provides the idea for further design of the experiment of TCM in the treatment of spleen deficiency and phlegm turbidity AS.The second study found that compared with the normal group,the model group intestinal epithelial cells denaturation,inflammatory infiltration,the situation after treatment reduced.Compared with the normal group,146 differential proteins were screened in the model group,including 99 up-regulated proteins and 47 down regulated proteins,and 8 representative proteins were concentrated in glycolipid metabolism pathway,bile acid secretion pathway and cell adhesion molecule pathway.Compared with the model group,189 differential proteins were screened out in the intervention group,including 70 up-regulated proteins and 119 down regulated proteins.Go enrichment analysis showed that these differential proteins were mainly enriched in binding,catalytic activity,transport activity,structural molecular activity and other biological processes;enriched in cell,cell components,organelles,cell membrane,extracellular area and other cell components;enriched in cell process,biological regulation,biological process regulation,metabolic process,stimulation response,positioning and other molecular functions.KEGG pathway analysis showed that the change of cell adhesion molecule pathway was the most significant,phagocyte,protruding vesicle cycle,amino acid biosynthesis,insulin secretion,PPAR signal pathway,fat digestion and absorption closely related to lipid metabolism,and bile acid secretion pathway significantly changed.In addition,oxidative phosphorylation and other pathway proteins closely related to energy metabolism also changed.Compared with the normal group,the activity of mitochondrial respiratory chain complex Ⅰ,Ⅱ,Ⅲ,Ⅳ and Ⅴ in the model group decreased significantly,and ATP synthetase decreased significantly(P < 0.05).Compared with the model group,the activitiesof mitochondrial respiratory chain complex Ⅰ and Ⅴ in the intervention group were significantly increased,and ATP synthetase was increased(P < 0.05).Compared with the normal group,the expression of IDH and Na+-K+-ATP in the model group increased significantly(P < 0.01),and the expression of ATP5 protein decreased significantly(P <0.01).Compared with the model group,the expression of IDH and Na+-K+-ATP decreased significantly in the intervention group(P < 0.01),and the expression of ATP5 protein increased significantly(P < 0.01).Conclusion:1."Spleen" is a concept of function in TCM.It is significant to treat AS from the spleen.The heart and the small intestine are the same as the main blood vessels,and the essence of the small intestine digestion and absorption and energy metabolism process is linked from the function of "the spleen is the main transporter",combined with the modern medical proteomics and the pathological relationship between mitochondrial energy metabolism and AS,the scientific hypothesis is made: the treatment of spleen deficiency and phlegm turbid AS by TCM of invigorating the spleen and removing phlegm and stasis may have an effect from the perspective of small intestine proteomics and mitochondrial energy metabolism.2.Jianpi Qutan Huayu decoction can improve the damage of small intestinal epithelial cells,and affect the expression of 189 proteins such as Cox 5B in the small intestine of AS Bama pig with spleen deficiency and phlegm turbidity.These proteins are mainly enriched in cell adhesion molecules,fat digestion and absorption,bile acid secretion,oxidative phosphorylation and other pathways.Jianpi Qutan Huayu decoction can improve the state of spleen deficiency and high fat by regulating the expression of these key pathways The digestive and absorptive function of the small intestine of Bama pig is abnormal,and then it can improve the AS of spleen deficiency and phlegm turbidity.3.Jianpi Qutan Huayu Chinese herbal formula may improve the activity of mitochondrial respiratory chain complex and ATPase,increase the content of I,V and ATP in small intestine tissue,regulate the expression of ATP5,IDH and Na+-K+-ATP protein,intervene the energy metabolism of small intestine mitochondria,improve the small intestine transport function under pathological state,and prevent and treat AS.