| Background:Based on the clinical experience of Traditional Chinese Medicine(TCM)and the theory of modern fluctuation effects,our subject has proposed the concept that multiple trace corxponents of Chinese medicine compound will affect the organism and produce overall effects after entering the body,and suggests the hypothesis of"multi-component biological effects model at effective threshold concentrations or extremely low concentrations." If these hypotheses are verified,a new perspective will be provided on the understanding of the complex life phenomena including the effects of prescription medicine in TCM.The general phenomenon of the biological effects of the coexistence of various components in TCM at extremely low concentrations will also be found.It will also have a revolutionary impact on the development of new drugs,including Chinese and western medicines.Objective:Based on the previous work basis of our subject,it is proposed that the four chemical components(puerarin,baicalein,berberine,and glycyrrhizic acid)and their compatibility involved in the 4 Chinese herbal medicines in Gegen Qinlian decoction will be used as characterizations of the prescription,taking the study of the mechanism of insulin resistance(IR)in the development of type 2 diabetes as the background,the important target organs/target cells of IR,hepatocytes(HepG2 cells),were selected as probing tools.After observing the effects of different concentrations of monomer components on the glucose metabolism of this IR cell model,molecular biological detection techniques were used to further investigate the anti-IR effect and mechanism of extremely low concentration compatibility under the effect threshold,in order to explore the biological effect model of compatibility of TCM compound at low concentrations of multiple components,to provide a new basis for the speculation of the principle of "multiple-component effects at very low concentrations" in the principle of effectiveness of TCM prescriptions,and to provide a molecular understanding of the mechanism of action at very low concentrations of multi-component formulations.Methods:(1)Safe concentration range of four monomer components in vitro:HepG2 cells in logarithmic growth phase were inoculated on 96-well plates.After culturing for 24 hours,the original culture medium was discarded,and final concentrations of 10-9,10-8,10-7,10-6,10-5,and 10-4mol/L puerarin,baicalein,berberine,and glycyrrhizic acid were added,the normal group was added with the same volume of culture medium;placed in a 37?,5%CO2 incubator for 48 hours.CCK-8 method was used to detect the proliferation activity of HepG2 cells,and the concentration range of each monomer component in vitro was obtained.(2)Methodology study of HepG2 IR cell model replication:HepG2 cells in logarithmic growth phase were seeded on 96-well plates.After being incubated:in a 37?,5%CO2 incubator for 24 hours,the culture medium was aspirated and replaced with a serum-free medium for 12 hours,and the medium was aspirated.The normal group was added with normal culture medium.The model group was added with a newly prepared medium containing 25mmol/L glucose and insulin with respective concentrations of 1×10-9 to 1 ×10-5mol/L,placed in 37 ?,5%CO2 incubator for 24 hours,48 hours and 72 hours.Using CCK-8 method and glucose oxidase(GOD-POD)method,the effect of each concentration of insulin on cell proliferation activity and glucose consumption was tested,and the optimal insulin concentration and action time were screened.According to the screening conditions,the cells were incubated for 24 hours,48 hours,and 72 hours in normal medium without insulin,and the stability of the cell IR model was investigated by measuring the glucose consumption of the cells.(3)Concentration-effect range of anti-IR effect of four monomeric components in vitro:HepG2 cells in logarithmic growth phase were taken and plated in 96-well plates and divided into normal group,model group and administration group.In the administration group,puerarin,baicalein,berberine,and glycyrrhizin acid were added at final concentrations of 10-11 to 10-5mol/L,and the same volume of 25mmol/L glucose cell culture medium was added to the normal group and the model group;placed in a 37 ?,5%C02 incubator for 24 hours.After 24 hours of drug treatment,the concentration-effect range of anti-IR of the four monomer components in vitro was examined by measuring cell proliferation activity and glucose consumption.(4)Anti-IR activity and effect mechanism of 4 monomer components in vitro:HepG2 cells were divided into normal group,model group and administration group.Different concentrations(10-6 and 10-5mol/L)of puerarin,baicalein,berberine,glycyrrhizic acid,and metformin(10-3mol/L)were added to the administration group,the normal and model groups were added to the same volume cell culture medium;placed in 37?,5%CO2 incubator for 24 hours.Detection indicators:1)Cell proliferation activity and glucose consumption;2)Glucose utilization:pyruvate kinase(PK)and glucokinase(GCK)activity;3)Gluconeogenesis:glucose 6 phosphatase(G6Pase),phosphoenol pyruvate carboxykinase(PEPCK)mRNA expression level;4)Glucose uptake:cellular insulin receptor(InsR)mRNA expression level;5)Related signaling pathways:phosphatidylinositol 3-kinase(PI3K),porotein kinase B(AKT),glucose transporter 2(GLUT2)mRNA expression and cell insulin receptor substrate 1(I RS1),phospho-phosphoinositide 3 kinase(p-PI3K),phospho-protein kinase B(p-AKT),GLUT2 protein expression level;6)Energy metabolism:cellular SIRT1I SIRT3 mRNA expression and adenylate-activated protein kinase(AMPK),peroxisome proliferator-activated receptor gamma coactivator-1 alpha(PGC-1?),mitochondrial transcription factor A(TFAM),nuclear factor-associated factor 2(NRF-2),carnitine palmitoyltransferase 1(CPT-1),forkhead transcription factor 1(FOXOl)and acetyl-coA carboxylase(ACC)protein expression levels.(5)Anti-IR effects and mechanism of different compatibility of four monomer components at the effect threshold concentration:(1)Anti-IR effect of four monomer combinations at different effect threshold concentrations:HepG2 cells were divided into normal group,model group,the 4 compatibility groups of the respective threshold concentrations(1/4,1/10,1/100,1/1000)and metformin(10-3mol/L)group.They were placed in a 37?,5%CO2 incubator for 24 hours.Detection indicators:cell proliferation activity and glucose consumption.(2)Anti-IR effects of different combinations of 1/100 effect threshold concentrations of 4 monomer components:HepG2 cells were divided into normal group,model group,puerarin(A,10-11mol/L)group,puerarin + baicalein(A+B,10-11+10-10mol/L)group,puerarin + baicalein + berberine(A+B+C,10-11+10-10+10-11mol/L)group,puerarin +baicalein + berberine + glycyrrhizic acid(A+B+C+D,10-11+10-10+10-11+10-10mol//L)and metformin(10-3mol/L)for 7 groups,each group of drugs containing considerable concentrations was added to the cell culture medium;the cell culture plates were placed in a 37?.,5%CO2 incubator for 24 hours.Detection indicators:cell glucose consumption;cell PK,GCK,and GLUT2 activity.(3)Anti-IR mechanism of low concentration formulations(1/100 and 1/10)at the threshold concentrations of 4 monomer components:HepG2 cells were divided into normal group,model group,and 1/100 low-concentration formula(A+B+C+D,10-11+10-10+10-11+10-10mol/L)group,1/10 low-concentration formula(A+B+C+D,10-10+10-9+10-10+10-9mol/L)group and metformin(10-3mol/L)group,each medium containing a considerable concentration of each drug was added to the medium and placed in a 37?,5%CO2 incubator for 24 hours.Detection indicators:1)Gluconeo genes is:cellular G6Pase,PEPCK mRNA expression levels;2)Glucose uptake:cellular GLUT2,InsR,PI3K,AKT mRNA expression and GLUT2,IRS1,p-PI3K,p-AKT protein expression levels;3)Energy metabolism:cellular SIRT1,SIRRT3 mRNA expression and AMPK,PGC-1?,TFAM,NRF-2,CPT-1,FOXO1,and ACC protein expression levels.Results:(1)Safe concentration range of four monomer components in vitro:different concentrations of puerarin,baicalein,berberine and glycyrrhizic acid all had certain inhibitory effects on the proliferation of normal HepG2 cells,and the four monomer components at the concentration of 10-9 to 10-5mol/L had no significant effect on the proliferation of HepG2 cells in vitro(P>0.05).(2)Methodology study of HepG2 IR cell model replication:in vitro culture conditions,25 mmol/L glucose combined with 10-6mol/L insulin was induced for 24 hours,and a stable and reliable HepG2 IR model in vitro could be successfully reproduced.This model can last for 48 hours.(3)Concentration-effect range of anti-IR effect of four monomeric components in vitro:puerarin,baicalein,berberine and glycyrrhizic acid in a certain concentration range had no significant effect on the proliferative activity of HepG2(P>0.05),but it had a promoting effect on the glucose consumption of the IR model to different degrees,and showed a "concentration-effect" relationship.The effective concentrations of puerarin and berberine ranged from 10-9 to 10-5mol/L,and the effective concentrations of baicalein and glycyrrhizic acid were both in the range of 10-8 to 10-5mol/L.(4)Anti-IR activity and effect mechanism of 4 monomer components in vitro:puerarin,baicalein,berberine,glycyrrhizic acid and metformin in certain concentration range significantly increased glucose consumption in HepG2 IR model cells(P<0.05 or P<0.01 or P<0.001),increased PK and GCK activity(P<0.05 or P<0.01 or P<0.001),up-regulated the mRNA expression levels of InsR,PI3K,AKT,GLUT2(P<0.05 or P<0.01)and the protein expression levels of IRS-1,p-PI3K,p-AKT,GLUT2(P<0.05 or P<0.01),down-regulated the mRNA expression levels of G6Pase,PEPCK(P<0.05 or P<0.01).In addition,puerarin,berberine,and metformin also involved the up-regulation of SIRT1 and SIRT3 mRNA expression(P<0.05 or P<0.01)and AMPK,PGC-1?,TFAM,NRF-2,and CPT-1 protein expression levels(P<0.05 or P<0.01),and the down-regulation of FOXO1,ACC protein expression level(P<0.05 or P<0.01)in model cells.(5)Anti-IR effects and mechanism of different compatibility of four monomer components at the effect threshold concentration:1)the 1/4,1/10,and 1/100 compatibility of the four monomer components at different threshold concentrations had different degrees of improvement in glucose consumption in the HepG2 IR cell model(P<0.05 or P<0.01).2)Several different combination formulas(A,A+B,A+B+C,A+B+C+D)of 1/100 of 4 monomer threshold concentrations had different degrees of improvement in glucose consumption,PK,GCK and GLUT2 activity in HepG2 IR model cells(P<0.05 or P<0.01 or P<0.001),of which the effect of the four monomer combination groups(A+B+C+D)was the best.3)The 1/100 and 1/10 low-concentration formulas of the four monomer components at the threshold concentrations all significantly up-regulated the expression of InsR,PI3K,AKT and GLUT2 mRNA in model cells(P<0.05 or P<0.01 or P<0.001)and IRS-1,p-PI3K,p-AKT,GLUT2 protein expression levels(P<0.05 or P<0.01),down-regulated the G6Pase,PEPCK mRNA expression levels(P<0.05 or P<0.01);at the same time,the 1/100 and 1/10 low-concentration formulas also up-regulated SIRTI,SIRT3 mRNA expression(P<0.05 or P<0.01)and AMPK,PGC-1?,TFAM,NRF-2,CPT-1 protein expression levels in model cells(P<0.05 or P<0.01),down-regulated the protein expression levels of FOXO1 and ACC(P<0.05 or P<0.01).Conclusion:(1)The four monomer components of puerarin,baicalein,berberine and glycyrrhizic acid have a wide range of biological activity against hepatic cell IR.Puerarin and berberine are 10-9 to 10-5mol/L,baicalein and glycyrrhizic acid are 10-8 to 10-5mol/L.(2)The anti-IR effects of puerarin,baicalein,berberine,and glycyrrhizic acid on improving the glucose uptake,glucose utilization,and inhibition of gluconeo genes is are involved in the regulation of IRS1/PI3K/AKT/GLUT2 signaling pathway;puerarin and berberine are also involved in regulating the AMPK/SIRT/PGC-1? signaling pathway,suggesting that the role of monomeric components may also involve multiple targets,multiple links,and multiple pathways.(3)The compatibility of 10-2 concentration(10-10 to 10-11mol/L)of puerarin,baicalein,berberine and glycyrrhizic acid at the threshold concentration 10-9 to 10-8 mol/L)still have certain anti-IR activity,among which the compatibility of all four kinds of monomers is the best,indicating that there is a certain synergistic effect among the components at extremely low concentrations.(4)The anti-IR mechanism of extremely low concentration formulations(1/100 and 1/10)at the threshold concentrations of 4 monomer components involves improving glucose uptake,glucose metabolism,and energy metabolism,and the regulation of IRS1/PI3K/AKT/GLU-T2 or/and AMPK/SIRT/PGC-1? signaling pathway.It is shown that the hypoglycemic effect of the four monomer composition formulas at extremely low concentrations involves multiple links and has a certain molecular basis.This study is based on the theory of complex systems to study the possible multi-component low-concentration effect model and its mechanism of action of the chinese herbal compound,and at the cellular level,it is reconfirmed that the multiple monomer formulations at the threshold concentrations have significant biological effects and exact molecular mechanism.The results of this study demonstrate that the "multi-component extremely low concentration-effect" model exists in the utility principle of TCM prescriptions,and it is of great scientific significance to reveal the basis of the TCM compound effect and its mechanism of action from a new perspective. |
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