The Mechanisms Exploration Of Nourishing Yin Benefiting Qi And Transforming Stasis Method On Reducing Glucose Fluctuation

OBJECTIVE: To explore the molecular mechanism of reducing blood glucose fluctuation in diabetic GK rats by using gene chip technology to explore the mechanism of nourishing yin,benefiting qi and transforming stasis based on the theory of "the homology of spleen and pancreas ".METHODS: After 4 weeks of adaptive feeding of 60 GK rats,50 5-month-old male SPF type 2 diabetic GK rats with a randomized glycemic value higher than 11.1 mmol/L were included in this experiment and randomly divided into 5 groups:model,the high-,medium-,and low-dose groups of Shenqi compound group(the following are referred to as the Shen-high,Shen-medium,and Shen-low groups respectively)and the western medicine group were fed with high-fat diet for 8 weeks after adaptive feeding for 4 weeks,establishing a model of diabetic macroangiopathy.Ten male 5-month-old wistar rats were used as blank groups and were given daily feed.There were 6 groups.At the same time,the model was given drug intervention.After entering the formal experiment,continuous administration for 8 weeks,high-,medium-,and low-dose Shenqi compound extracts were intragastrically administrated in the Shen-high group,Shen-medium group,and Shen-low group respectively,and the western medicine group was given intragastric administration of sitagliptin suspension.The blank group and model group were given normal saline irrigation.During the drug intervention,the general state of the rats was observed,and the amount of gavage was adjusted according to the change of body weight.The blood glucose was measured at the time of 8:00,10:00,14:00,16:00,and 18:00 within one day of every week.The condition and the blood glucose fluctuations in each group of rats were measured using the mean daily blood glucose level(MBG),the standard deviation of the daily average blood glucose level(SDBG),the maximum blood glucose level fluctuation range(LAGE,the difference between the highest and lowest daily blood sugar levels).The MBG,SDBG,and LAGE of rats in each group were counted after 4 weeks and 8 weeks.Eight weeks later,blood samples were collected,and cholesterol(TC)and triglyceride(TG)were detected using a Model 7710 automatic biochemical analyzer.Serum insulin,glucagon,GLP-1 were measured by radioimmunoassay and ELISA;Serum C-reactive Protein(CRP)levels were measured by double-antibody sandwich ELISA;HE Staining and light microscopy were performed to observe the histopathological morphology of the abdominal aorta and pancreas in each group.The best-selected Shenqi compound group,model group,blank group,and western medicine group were selected.Each group received 3 rats with 50-100 mg of pancreas.RNA was extracted and differential gene detection was performed on all samples using Agilent 4*44k genome-wide expression profiling microarray.Differential genes were enriched with GO and Pathway,and PCR was performed to verify the key differential genes Id2,Usp2,Pik3r1,and Ddit4.RESULTS: After drug intervention,the mental state and coat color of each group were better than those of the model group.GK rats drink more water.In the fourth week of experiment,the water intake of the Shen-high group and the western medicine group was lower than that of the model group.In the eighth week of the experiment,the food intake of the Shen-medium group was lower than that of the model group,and the drinking water of each group was lower than that of the model group;Compared with the model group,the TC in the Shen-high,Shen-medium,and western medicine groups all reduced;Each-dose of Shenqi compound group and the western medicine group can reduce the TG level.Compared with the western medicine group,the TG of the Shen-low group is higher than the western.-medicine group.Compared with the model-.group,the INS of the western medicine group was higher than the model group,the GC of the western medicine group and the Shen-high group was lower than the model group,and the GC of the western medicine group was lower than the other groups;Compared with the model group,the CRP levels of each group in rats(except for the Shen-low group)were lower than those in the model group.Compared with the model group,the CLP-1 in each group was higher than that in the model group,while the GLP-1 levels in the high and low groups were lower than the western medicine group.Blood glucose fluctuations: MBG,SDBG,and LAGE of GK rats in each group were higher than normal wistar rats in the 4th week.Compared with the model-group,the MBG of the Sengao group was lower than that of the model group.The SDBG of the middle group and western medicine group was lower than that of the model group,and the LAGE of each group of Shenqi compound and Western medicine group was lower than that of the model group.In the eighth week of the experiment,compared with the model group,the MBG,SDBG,and LAGE in each group were lower than those in the model group(except for the SDBG of Shen-low group).Compared with the western medicine group,the SDBG and LAGE in each dose group of Shenqi compound were higher than those in the western medicine group;Pathomorphological results: Shen-high,Shen-medium and western group were more effective in improving the pathological morphology of the main abdominal artery.Shen-medium,Shen-high group were more significant improvement in the pathological morphology of the pancreas,followed by the western and Shenlow group.Gene chip test results: differential gene screening: there were 349 common differential genes in the model group vs normal group,of which 128 were up-regulated and 211 were down-regulated.There were 77 differential genes in the Shen-medium vs model group,of which 54 were up-regulated and 23 were down-regulated.There were 95 differentially expressed genes in the western group vs model group of western medicine group,of which 46 were up-regulated and 49 were down-regulated.GO analysis of differential genes.The differential genes in the model group and the VS blank group involve biological processes that can be summarized as metabolic processes,circadian rhythms,cell proliferation,cell secretion,leukocyte adhesion,cell migration,response to light stimuli,etc.The differential genes in the Shen-medium vs model group involved biological processes including circadian rhythms,cell proliferation,cell migration,cell adhesion,negatively regulated tumor necrosis factor-mediated signaling pathways,cellular response to stimulation,wound healing,and the negative regulation of TOR signaling,regulation of phosphatidylinositol 3-kinase activity,apoptotic signaling pathways.Western medicine vs model group differential genes involved in biological processes such as circadian rhythm,cell migration,cell adhesion,cell proliferation,regulation of wound healing,regulation of inflammatory response,regulation of homeostasis,insulin secretion and other biological processes.Pathway analysis of differential genes: The main pathways involved in the model group and the normal group were arginine and proline metabolism and metabolic pathways.The main pathways involved in the vs model group were pancreatic cancer,m TOR signaling pathway,renal cell carcinoma,bacterial invasion epithelial cells,VEGF signaling pathway,small cell lung cancer,Erb B signaling pathway,Fc?R mediated phagocytosis,Gn RH signaling pathway,regulating actin cytoskeleton,T cell receptor signaling pathway,leukocyte transendothelial migration,local adhesion,etc.Western medicine vs model group differential genes involved in the main pathway for arginine and valine metabolism,salivary secretion,Gn RH signal path.Combined with gene chip pathway and GO functional enrichment of differential gene analysis results,we found that the key pathway in the Shen-medium vs model group was the m TOR signaling pathway,and the differential genes were: Pik3r1 and Ddit4,both of which were up-regulated;The Shen-medium group vs the model group and the western medicine group vs model group all involved the GO items which were circadian rhythms.The key genes for enrichment were Id2 and Usp2,both showing down-regulation of Id2 expression and up-regulation of Usp2(P<0.05);the key difference gene Id2,Usp2,Pik3r1,and Ddit4 were verified by quantitative PCR.In conclusion,in the reference Shen-medium vs model group,Id2 expression is down-regulated,and Pik3r1,Usp2,and Ddit4 are all up-regulated.After verified by PCR,the expression trend was consistent and statistically significant.Conclusion: The method of nourishing yin,invigorating qi and transforming stasis can improve the function of islet cells,reduce blood glucose fluctuation,and delay macrovascular disease in diabetic GK rats;The method of nourishing yin,invigorating qi and transforming stasis can upregulate the expression of genes Pik3r1 and Ddit4 and affect m TOR signaling pathway.Inhibition of m TOR,activation of autophagy on protection of islet ? function;Blood glucose fluctuations in diabetic GK rats correlate with circadian rhythm gene expression in pancreatic tissue.The method of nourishing yin,invigorating qi and transforming stasis may play a role in stabilizing blood glucose fluctuations by regulating the expression of Id2 and Usp2 genes in the pancreas.Comparative analysis of gene chip results between model group/blank group,Shenqi compound group/model group,sitagliptin group/model group,the up-regulation of gene expression between Shenqi compound group/model group are more than other groups,and the enrichment of the pathways are also more than other groups,reflecting the multi-targeted,multi-pathway treatment characteristics of Shenqi compound.