Effect And Mechanism Of "Purging Fire And Reinforcing The Healthy Qi" Therapy On Dedifferentiation Of ? Cells In Type 2diabetes Mellitus Based On PI3K-Akt-FoxO1 Pathway

Objective1.Explore the thought of“fire-heat”in Shanghan Zabing Lun to provide theoretical support for the treatment of type 2 diabetes mellitus(T2DM)with pure Chinese medicine created by Professor Li Saimei.2.Study the efficacy and safety of pure Chinese medicines of“purging fire and reinforcing the healthy qi”method based on“fire-heat theory”created by Professor Li Saimei in the treatment of newly diagnosed T2DM.3.Predict the potential mechanism of Han Tang Ping(HTP),the core prescription of“purging fire and reinforcing the healthy qi”method,on improving islet?cell lesion in T2DM.4.Study the effect of HTP on dedifferentiation of islet?cells in db/db mice.5.According to the prediction results,verify the mechanism of HTP in improving?cell dedifferentiation induced by glucolipotoxicity.Methods1.Mine the thought of“fire-heat”in the prescriptions from Shanghan Zabing Lun using the Ancient and Modern Medical Cases Cloud Platform.We collected the prescriptions related to“fire-heat”in Shanghan Lun and Jingui Yaolve,then built the database of“fire-heat”prescriptions and syndromes,and used the data mining analysis function module of the Ancient and Modern Medical Cases Cloud Platform to carry out frequency statistics of single medicine,qi,flavor and meridian entry statistics,efficacy statistics,correlation analysis,cluster analysis,community analysis and complex network analysis.2.The efficacy and safety of pure Chinese medicines of“purging fire and reinforcing the healthy qi”method based on“fire-heat theory”created by Professor Li Saimei in the treatment of newly diagnosed T2DM.The newly diagnosed T2DM cases treated with pure Chinese medicine by Professor Li Saimei were collected.The data of glycated hemoglobin(Hb A1c),oral glucose tolerance test(OGTT),insulin release test,blood lipid,uric acid,liver and kidney function and adverse reactions were extracted before treatment and 3 months and 6 months after treatment.The main evaluation indexes:Hb A1c,Homeostasis Model Assessment 2-insulin resistance(HOMA2-IR),HOMA2-?;secondary evaluation indexes:area under OGTT curve(AUC_OGTT),area under insulin curve(AUC_INS),blood lipid and uric acid levels,adverse reactions,etc.3.Predict the potential mechanisms of HTP on improving islet?cell lesions in T2DM based on systems pharmacology and bioinformatics.The compounds and targets of HTP,a core prescription of“purging fire and reinforcing the healthy qi”method,were searched using systems pharmacology.The genes related to T2DM islet?cell lesion were identified by bioinformatics.Then the overlapped genes were considered as the target genes of HTP in the treatment of diabetic islet?cell lesion.Then,the intersection targets were used for PPI network and Chinese medicines-compounds-targets network construction,Gene Ontology(GO)function enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment,so as to predict the potential mechanism of HTP on improving islet?cell lesions in T2DM.4.Effect of HTP on dedifferentiation of islet?cells in db/db mice.50 db/db mice aged 6-8 weeks were randomly divided into the model group,the metformin group and the HTP low,medium,and high dose groups,with 10 C57 mice as the normal group.The 24h water intake,food intake,urine output,Lee's index,intraperitoneal glucose tolerance test(IPGTT),intraperitoneal insulin tolerance test(IPITT)and organ index of fat were observed.The pathological changes of pancreas were observed by HE staining.The protein expression levels of?cell maturation markers(INS,Maf A,Pdx-1)and dedifferentiation markers(Nanog,Ngn3)were detected by western blotting.5.Explore the mechanism of HTP in improving?cell dedifferentiation induced by glucolipotoxicity based on PI3K-Akt-FoxO1 pathway.Cell Counting Kit-8(CCK8)method and glucose-stimulated insulin secretion(GSIS)experiment were used to construct the suitable high glucose and high fat culture environment and intervention time for MIN6 cells.HTP-contained serum was prepared,and the suitable dilution times of HTP-contained serum were explored with the same methods.Then the expression levels of?cell maturation markers(INS,Maf A,Pdx-1),dedifferentiated progenitor cell markers(Ngn3,Nanog)and PI3K-Akt-FoxO1 pathway key proteins(p-Akt,Akt,p-Fox O1,Fox O1)in the normal group,the high glucose and high fat model group,the HTP medicated serum group and the PI3K inhibitor group were detected by western blotting.Results1.Mine the thought of“fire-heat”in the prescriptions from Shanghan Zabing Lun using the Ancient and Modern Medical Cases Cloud Platform.The“fire-heat”syndromes treated by the prescriptions in Shanghan Zabing Lun could be divided into“excess fire”,“Yin fire”,“mixed fire”,“deficiency fire”and“floating fire”.Under the guidance of the concept of syndrome differentiation and treatment of“observing the pulse disease,knowing what the disease is,and treating it according to the syndrome differentiation”,the qi,flavor and efficacy of the“fire-heat”prescriptions mainly focus on bitter-cold,purging fire and eliminating pathogenic factors,and pay attention to sweet-warm,unstopping-tonifying and reinforcing the healthy qi.Meridian entry was all over the six meridians,emphasizing lung,spleen,gastrointestinal,liver,gallbladder,kidney.The drugs are closely related,and the combination is accurate and appropriate,which contained the treatment ideas of treating cold with heat,treating heat with cold,combined use of cold and heat,reinforcing the healthy qi and eliminating the pathogenic factors.The ultimate goals were to remove“fire-heat”,strengthen healthy qi and heal disease and syndrome.2.The efficacy and safety of pure Chinese medicines of“purging fire and reinforcing the healthy qi”based on“fire-heat theory”created by Professor Li Saimei in the treatment of newly diagnosed T2DM.A total of 47 cases were included.After 3 or 6 months of treatment,Hb A1c,AUC_OGTT,TG and LDL-C were significantly decreased(P<0.001,P<0.01,P<0.05);HOMA2-?was significantly increased(P<0.01,P<0.001);compared with 3 months of treatment,Hb A1c and AUC_OGTT were significantly decreased(P<0.05).The effective rate was 91.7%,and there was no obvious adverse reaction.3.Predict the potential mechanisms of HTP on improving islet?cell lesions in T2DM based on systems pharmacology and bioinformatics.Its potential mechanisms were complex.There were 71 active components,63 potential targets,701 biological processes,69 cell components,77 molecular functions and 110signaling pathways including Fox O pathway and PI3K-Akt pathway,which were involved in the potential mechanisms of HTP in improving diabetic islet?cell lesions.4.Effect of HTP on dedifferentiation of islet?cells in db/db mice.Compared with the normal group,the 24h water intake,food intake,urine output,?AUC_IPGTT and organ index of fat in the model group,the metformin group and the HTP low,medium and high dose groups were significantly increased,and?AUC_IPITT was significantly decreased(P<0.001);compared with the model group,changes in these indicators were significantly reversed in the metformin group and the HTP low,medium and high dose groups(P<0.01,P<0.001,P<0.05).Compared with the normal group,the Lee's index in the model group and the HTP low and medium dose groups were significantly increased(P<0.01,P<0.001,P<0.05);compared with the model group,it was significantly reduced in the metformin group and the HTP medium and high dose groups(P<0.05).Compared with the normal group,the other groups showed obvious pancreatic lesions,including islets hyaline degeneration,islets atrophy,vacuolar degeneration,lobular duct hyperplasia,pancreatitis,necrosis and pancreatic atrophy.In the model group,the number of lesions was the largest,the scope was widest,the forms was the most various and serious.After the treatment with metformin or HTP,the pancreatic lesions could be markedly alleviated.Compared with the normal group,the levels of INS,Maf A and Pdx-1 in the model group were significantly decreased(P<0.001);compared with the model group,these proteins were significantly increased(P<0.001)in other groups;compared with the normal group,Nanog and Ngn3 in the model group were significantly increased(P<0.01);compared with the model group,these proteins in the metformin group and the HTP low,medium and high dose groups were significantly decreased(P<0.01,P<0.001,P<0.05).5.Explore the mechanism of HTP in improving?cell dedifferentiation induced by glucolipotoxicity based on PI3K-Akt-FoxO1 pathway.CCK8 and GSIS experiments confirmed that 6 g/L glucose+0.2 mmol L PA for 6 hours had little effect on MIN6 cell survival rate,but had strong inhibitory effect on insulin secretion.Because the dedifferentiated islet?cells have the characteristics of living cells and no insulin secretion function,combined with cell survival rate and GSIS experiment,we chose this condition as the high glucose and high fat model to induce the dedifferentiation of islet?cells.In the same way,it was found that the 20 folds dilution of HTP-contained serum had little effect on the survival rate of?cells under glucolipotoxicity,but had strong effect on promoting insulin secretion.Thus,we chose this dilution as the intervention concentration of HTP-contained serum.In the expression of INS,Maf A and Pdx-1:compared with the normal group,INS,Maf A and Pdx-1 in the model group and the inhibitor group were significantly reduced(P<0.001,P<0.01);compared with the model group,these proteins in the HTP-contained serum were significantly elevated(P<0.001,P<0.01);compared with the HTP-contained serum,the expression levels of these proteins in the inhibitor group were significantly reduced(P<0.001,P<0.01).In terms of Nanog and Ngn3 proteins expression:compared with the normal group,the model group,the HTP-contained serum group and the inhibitor group were significantly elevated(P<0.001);compared with the model group,the HTP-contained serum group and the inhibitor group were significantly reduced(P<0.001,P<0.01);compared with the HTP-contained serum group,the inhibitor group was significantly elevated(P<0.01).In PI3K-Akt-FoxO1 pathway key proteins expression:p-Akt/Akt:compared with normal group,the model group and inhibitor group were significantly decreased(P<0.001);compared with model group,the HTP-contained serum group and the inhibitor group were significantly increased(P<0.001);compared with the HTP containing serum group,the inhibitor group was significantly decreased(P<0.001).p-Fox O1/Fox O1:compared with the normal group,the model group,the HTP-contained serum group and the inhibitor group were significantly decreased(P<0.001,P<0.01);compared with the model group,the HTP-contained serum group and the inhibitor group were significantly increased(P<0.001);compared with the HTP-contained serum group,the inhibitor group was significantly decreased(P<0.001).ConclusionThe pure Chinese medicines of“purging fire and reinforcing the healthy qi”method created by Professor Li Saimei is safe and effective in the treatment of T2DM.Her thought of“fire-heat”has a deep root in Shanghan Zabing Lun,and the potential mechanism is complex.One of the possible mechanisms may improve the dedifferentiation of islet?cells via activating PI3K-Akt-FoxO1 pathway.