| Objective:In the study,GK rats are selected as the model animals of diabetic macrovascular lesions to study the effect of Shenqi compound,which is the representative formula of nourishing Yin benefiting Qi and activating blood method,on GK rats’ macroangiopathy.Application of gene expression profile chip technology to explore the molecular mechanism of nourishing Yin benefiting Qi and activating blood method intervention diabetic macrovascular lesions,from different m RNA expression and TLR signaling pathways to explore the nourishing Yin benefiting Qi and activating blood method through blocking "metabolic memory" and regulating immune-inflammatory microenvironment approach to prevention and control of the molecular mechanism of diabetic vascular lesions.Methods:The first part: The study on the effect of nourishing Yin benefiting Qi and activating blood method on the macrovascular lesions in GK rats.100 7-8-week-old male spontaneous type 2 diabetic GK rats with fasting blood glucose ≥ 11.1mmol/l were fed with a high-fat diet for 4 weeks to maintain hyperglycemia state to simulate the "metabolic memory" process of hyperglycemia and establish a model of diabetic macroangiopathy.After the model was established successfully,100 experimental rats were randomly divided into 5 groups,which were the model group,the western medicine group,the shengao group,the shenzhong group and the shendi group,20 GK rats in each group and 20 Wistar rats as a blank group.Western medicine group was given metformin gavage intervention,Shengao group,Shenzhong group and shendi group were given Shenqi compound high,medium and low dose gavage intervention,model group and the blank group were given distilled water gavage intervention for 12 weeks.During the experimental period of gavage intervention,the general condition of six groups of rats was closely observed,and the body weight and fasting blood glucose were measured every week.After the experiment,the serum Chol level was detected;The serum TLR4,IFN-γ,IL-4 levels were detected by ELISA;The HE staining was used to observe the morphological changes of thoracic aorta;The apoptosis of thoracic aorta was observed by TUNEL staining.The second part: The study of the effect of nourishing Yin benefiting Qi and activating blood method on the m RNA expression profile of GK rats.The methods of modeling,grouping and administration were the same as the first part.At the end of the intervention,gene chip technology was used to detect the m RNA expression profile of four samples randomly selected from each group of thoracic aorta,and protein interaction network analysis,go enrichment analysis and KEGG enrichment analysis was used to analyze the different m RNA related to immune inflammation.Results:The first part: 1.The model of diabetic macroangiopathy in GK rats was established successfully: Compared with the Wistar rats in the blank group,GK rats in the model group were depressed in spirit,slow in response,sluggish in movement,slow in weight increase,and there were important risk factors of diabetic macroangiopathies,such as hyperglycemia,hyperlipidemia and immune inflammation disorder;HE staining of thoracic aorta showed severe vascular endothelial damage;TUNEL staining showed obvious apoptosis and increased apoptosis rate,all of which were in line with diabetic macroangiopathy Characteristics of lesions.2.The effect of nourishing Yin benefiting Qi and activating blood method on diabetic macroangiopathy in GK rats was definite: After the treatment of Shenqi compound,which is a representative formula of nourishing Yin benefiting Qi and activating blood method,the mental state of rats in each group of Shenqi compound group gradually became better,blood glucose and lipid levels decreased,TLR4 expression decreased,the expression level of pro-inflammatory cytokines IFN-γ decreased,the expression level of anti-inflammatory cytokines IL-4 increased,vascular endothelial injury recovered to near normal,the apoptosis of thoracic aorta tissue decreased,from diabetic macrovascular lesions important risk factors of blood sugar,blood lipid and immune inflammation medium level of change,as well as the pathology and apoptosis of the thoracic aorta all to reflect the effective intervention of nourishing Yin benefiting Qi and activating blood method on the diabetic macrovascular disease.3.Nourishing Yin benefiting Qi and activating blood method could regulate the immune balance of Th1/Th2 cells,reduce inflammation,inhibit cell apoptosis,and block the metabolic memory by reducing the level of TLR4 and regulating the expression of Th1 and Th2 cytokines,so as to prevent and treat diabetic macrovascular lesions: After the treatment of Shenqi compound,the expression of TLR4,IFN-γ and the level of IFN-γ/IL-4 in serum decreased,the expression of IL-4 increased,and the apoptosis of thoracic aorta tissue decreased,which indicated that the method of nourishing Yin benefiting Qi and activating blood method could shift the immune balance of Th1/Th2 to Th2 cells,reduce the inflammatory reaction,inhibit cell apoptosis,and block the metabolic memory,so as to prevent and treat diabetic macrovascular lesions.The second part: 1.Nourishing Yin benefiting Qi and activating blood method could widely regulate the differential m RNA expression in the thoracic aorta of GK rats: Compared with the Wistar rats in the blank group,the differential m RNA expression in the thoracic aorta of GK rats in the model group was obvious,which determined the differential expression of diabetic macroangiopathy in the pathological morphology of GK rats;Compared with the model group,the difference m RNA in the Shengao group and the Shenzhong group was mainly regulated above,and in the Shengao group the regulation trend was more obvious,and the difference of m RNA in the low group was mainly under regulation.2.Nourishing Yin benefiting Qi and activating blood method could widely regulate the expression of immune inflammation-related differential m RNA in the thoracic aorta of GK rats: Compared with the Wistar rats in the blank group,the expression of the immune inflammation-related differential gene in the model group was obvious,which suggested that the immune inflammation process might be involved in diabetic macroangiopathy;Compared with the model group,the difference m RNA related to immune inflammation in the high and middle groups were mainly modulated,and the up-regulation trend of Shenqi compound was more obvious in Shengao group;The downregulation of differential m RNA was the main one in shendi group;In the top ten immunoinflammatory related differential m RNA,the reverse regulated differential m RNA of Shenqi compound was IRF7,MX2,CCL21,ADRB2.3.Nourishing Yin benefiting Qi and activating blood method could regulate the expression of the key genes of protein interaction network in GK rats: The analysis of protein interaction network in rat thoracic aorta after the treatment of Shenqi compound showed that the network nodes of the differential genes IL6,SOCS3,CCL2 and CXCL1 were the highest,and these genes might be the key genes of Shenqi compound regulating the process of immune inflammation in diabetic rats.4.Nourishing Yin benefiting Qi and activating blood method could regulate the biological process closely related to different m RNA immune inflammation: Go enrichment analysis showed that compared with the model group,Shenqi compound could reverse regulate the biological process related to different m RNA MX2,IRF7,ADRB2 and CCL21 related to immune inflammation,as well as the biological process related to key genes IL6,CCL2 and CXCL1 related to immune inflammation.5.Nourishing Yin benefiting Qi and activating blood method could regulate the TLR signal pathway and NLR signal pathway: KEGG pathway enrichment analysis showed that the most significant(Q-value < 0.05)signal pathway involved in the regulation of immune inflammation-related differential m RNA by Shenqi compound was TLR signal pathway and NLR signal pathway.6.Nourishing Yin benefiting Qi and activating blood method could affect the expression of TLR signal pathway-related differential genes: The statistical results of TLR signal pathway differential genes showed that there were four significant difference genes IRF7,IL6,Fos and nfkbia in Shengao group compared with the model group,speculated that the nourishing Yin benefiting Qi and activating blood method might affect the TLR signal pathway by regulating the expression of IRF7,IL6,Fos and nfkbia genes,so as to prevent and treat diabetic macrovascular Pathological changes.Conclusion:The nourishing Yin benefiting Qi and activating blood method can improve the general state of GK rats with diabetic macroangiopathy,reduce blood glucose,improve lipid metabolism disorder,adjust Th1/Th2 immune balance,reduce inflammatory response,protect vascular endothelial damage,and block "metabolic memory".Its mechanism may be through regulating the biological functions closely related to immune inflammation and TLR signaling pathway,NLR signaling pathway and Irf7,Mx2,Ccl21,Adrb2,Il6,Socs3 and other genes,so as to promote the repair of damaged vascular endothelial cells in the early stage of diabetic macroangiopathy,in which the regulation of TLR signaling pathway is to adjust the Th1/Th2 immune balance,reduce inflammation,inhibit cell apoptosis,and block the metabolic memory of diabetic macroangiopathy.It embodies the TCM advantages of Shenqi compound,which is the representative formula of nourishing Yin benefiting Qi and activating blood method,in the whole treatment,in the combination of prevention and treatment,and in the mild Fire supplementing Qi to reinforce the healthy and eliminate the pathogenic. |
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