| Purpose:Explore the potential targets and action mechanisms of Shen Qi formula on type 2 diabetes(T2DM)treatment based on network pharmacology;examine the effectiveness of Shen Qi formula on islet microcirculation improvement through experiments.Methods:1.First,proteins and targets of Shen Qi formula are collected using TCMSP database.Target names are simplified on the basis of Uniprot database.Network pharmacology studies on Shen Qi formula are performed using Cytoscape3.6.1 with the aim of discovering network properties between molecular and target.Second,diseaserelated targets are obtained through CTD、TTD and Drug Bank databases.Common targets for proteins and diseases are screened out to perform associate analysis between proteins and targets as well as between diseases and targets.Third,protein–protein interactions(PPI)are investigated on the basis of STRING 11.0 database.At last,KEGG analysis is conducted to obtain the metabolic pathway of Shen Qi formula on T2 DM treatment.2.70 Sprague-Dawley(SD)rats were fed adaptively for one week.Diabetes model was prepared using streptozotocin(STZ).Rats with type 2 diabetes were randomly divided into 5 groups,i.e.,model group(M),high-dose group(SH),middle-dose group(SM),low-dose group(SL),Gehuazhi group(G).Thereafter,subcutaneous injection of ultra-low-acting insulin 3U and intraperitoneal injection of 250g/L glucose solution 0.38g/kg·d were given at regular intervals for 8 weeks and 2 days per week to prepare diabetic blood glucose fluctuation model.10 normal SD rats were further selected as a blank control group(C).Each group of rats was under daily drug intervention.During the intervention period,statistics on rates’ living condition(e.g.,weight,food intake,water consumption)were recorded daily.The blood glucose level was measured once a week.Blood glucose fluctuation indexes(e.g.,MBG,SDBG,LAGG)were calculated based on these statistics.Rates were killed after 8-week intervention to obtain final intervention results.Blood samples were collected from abdominal aorta.Serum insulin,renin,angiotensin II(Ang II),and aldosterone(ALD)were detected using ELISA method.Pathological change of pancreatic tissue was recorded using HE staining.The number and density of islet micro-vessels and the thickness of islet microvascular wall were also recorded.The number of islets α and β cells were detected using immunohistochemical method.Islet cell apoptosis was detected using TUNEL method.High-throughput sequencing method is used for Affymetrix WT expression chip sequencing of islet micro-vessels.GO and KEGG analyses are performed based on David database.Expression levels of ACE2,IRS-1,and AKT1 m RNA for islet tissue were obtained using QPCR method.Expression levels of proteins ACE2,MAS1,IRS-1,and pan-AKT were acquired using Western Blot method.Results:1.Network pharmacology results(1)A total number of 152 proteins and 278 targets is obtained for Shen Qi formula.26924 pairs of disease and target exist for T2 DM.A number of 261 common targets of disease and protein is obtained.Associate analysis between protein and target shows that NOS and AKT are major target groups,therefore implying that they are closely related to metabolic and inflammation-related pathways of T2 DM.(2)KEGG analysis on common targets show that 182 pathways exist for Shen Qi formula to heal T2 DM.52 pathways are closely related to T2 DM treatment according to literature.They include oxidative stress(AGE-RAGE pathway),inflammatory response(IL-17 pathway,TNF pathway),apoptosis,and insulin resistance.(3)Comparison between literature and aforementioned common targets and pathways shows that about 35 protein targets are involved in islet microcirculation.These protein targets include protein kinase B(AKT),matrix metalloproteinase(MMP),mitogen-activated protein kinase(MAPK),and renin angiotensin(ACE2,ANG2,ACE)families.ACE2,Mas,IRS-1,and AKT were selected as key regulatory targets for QPCR and WB experimental verification.2.Experiment results(1)Islet pathological results show that rats in SH,SM and SL groups have better conditions(e.g.,bright coat color and good mental state)than other groups.The weight of rats in groups M and G decreased continuously after modeling.However,the weight increases after decreasing for SH,SM,SL groups(p<0.01,p<0.05).Rats show increased food intake and water consumption after modeling process.However,rats in SH,SM and SL groups show reduced polyphagia and polydipsia comparing with M group.The magnitude of islet injury for different groups follows the order: M>SH>G>SM>SL>C.(2)Results of islet endocrine cell and glucose-regulating hormone show that the impact of modeling process on the number of islet α cell is not significant(p>0.05),while the impact on the number of islet β cell is significant(p<0.01).After the treatment,the number of islet β cell for rats in SL group increases significantly comparing with M group(p<0.05).Islet cell apoptosis for SH,SM,and SL groups is improved significantly comparing with M group(p<0.05).The insulin level in SH group also shows improvements comparing with M group after treatment(p<0.05).(3)Analysis on the component structure of islet microcirculation shows that the treatment significantly improves rats’ islet microvascular wall thickening for SH,SM,and SL groups comparing with M group.Also,comparing with M group,the number of islet micro-vessels in SM group increases significantly(p<0.05)as well as the islet micro-vessel density in SL and SM groups(p<0.05).(4)Results of Renin angiotensin system(RAS)hormone show that modeling process significantly decreases the serum renin(Renin)level(p<0.05).However,the treatment has insignificant effect on its improvement(p>0.05).Ang II decreases significantly in SH,SM,and SL groups after treatment comparing with M group(p<0.01).The treatment also significantly lowers the serum aldosterone level in SL group comparing with groups M and G(p<0.05).(5)Affymetrix WT expression chip sequencing and bioinformatics analysis on islet microvascular showed that different groups express genes differently at a significant level:(1)Compared with C group,M group obtained a total number of 183 different genes,of which 59 genes are up-regulated(32.24%)and 124 genes are down-regulated(67.76%);GO analysis obtained 99 items.44 items(44.4%)relate to biological processes in different ways,such as involvement of insulin secretion in cell response to glucose stimulation,G-protein coupling body signal pathways;KEGG analysis obtained a number of 16 items,including starch and sucrose metabolism,reninangiotensin system,carbohydrate digestion and absorption,endocrine and resorption of other calcium-regulated factors,type I diabetes,metabolic pathways.(2)Compared with M group,SL group has a number of 321 differential genes,of which 239 genes(74.45%)are up-regulated and 82 genes(25.55%)are down-regulated;GO analysis obtained 93 items.59 items(63.4%)pertain to biological processes,such as G proteincoupled receptor signaling pathway,angiogenesis,and angiogenesis;KEGG analysis obtained a number of 9 items,including ribosomes and metabolic pathways.(3)Compared with M group,G group has a number of 301 different genes,of which 183 genes(60.80%)are up-regulated and 118(39.20%)genes are down-regulated;GO analysis obtained a number of 74 items,including 50 items of biological process.These biological processes pertain to Wnt signaling pathway,calcium regulatory pathway,and others.KEGG analysis obtained a number of 15 items,including Wnt signaling pathway,m TOR signaling pathway,Hippo signaling pathway,and others.(6)Analysis shows that expression levels of ACE2 and IRS-1 m RNA in SL groups are increased comparing with M group(p<0.01,p<0.05),while the expression levels of AKT1 m RNA is not significant(p>0.05).However,Analysis on major proteins shows that expression levels of ACE2,Mas1,IRS-1and pan-AKT m RNA in SM and SL groups are increased significantly comparing with M group(p<0.01,p<0.05).Conclusions:1.Shen Qi formula has the characteristic of being multiple proteins,targets and action mechanisms.It prevents and treats T2 DM by controlling multiple target proteins and pathways.2.Shen Qi formula can improve islet microcirculation in rats with diabetic blood glucose fluctuation.It improves diabetes symptoms of rats with diabetes glucose fluctuation,such as polydipsia,polyphagia,weight loss and pathological morphology of islets.It improves islet microcirculation by reducing vascular damage of RAS hormone and restoring islet microvascular blood supply.3.The pathogenesis of islet microcirculation disorders is characterized by multiple genes and multiple pathways.Renin-angiotensin system could be an important part involving in islet microcirculation.One possible action mechanism is that Shen Qi formula stimulates RAS bypass pathways(ACE2/Mas axis)and promotes the expression levels of IRS-1 and AKT positively. |
