The Mechanism Of Huanglian-renshen-decoction,a Fixed Mixture Of Traditional Chinese Medicine,on Regulating Pancreatic Cells Transcription Factors To Improve Glucose Metabolism

Section 1 The mechanism of Huanglian-Renshen-Decoction on reulating pancreatic cells transcription factors to improve the glucose metabolism in db/db miceObjectiveTo explore the mechanism of Huanglian-Renshen Decoction(HRD)on regulating pancreatic cells transcription factors to improve the glucose metabolism.MethodsForty 7-8 week-old db/db(BSK)mice were randomly assigned to the following four groups: model,low dose HRD(LHRD),high dose HRD(HHRD)and saxagliptin(SAX).Additionally,10 db/m mice were assigned to control group.The experimental mice were administered 3.03g/kg/d and 6.06g/kg/d of HRD in the LHRD and HHRD groups,respectively,and 10mg/kg/d saxagliptin in the SAX group for 8 weeks.The control and model groups were supplied with distilled water.The fasting blood glucose(FBG)from tail vein,body weight and 24 h food intake were detected weekly.In the end of experimental stage,intra-peritoneal glucose tolerance test(IPGTT)and intra-peritoneal insulin tolerance test(IPITT)were performed in different day.After the intervention,the pancreas and blood were collected and tested.Elisa kit was used to test the serum insulin level.The insulin,glucagon,Nkx6.1,Pdx1,Ki67 and Caspase12 in islet were tested with immunofluorescence.Western Blotting examined the protein levels of Cleaved caspase3/Caspase3,Bax/Bcl2 and Ngn3.In addition,the immunohistochemistry tested the Cleaved Notch1 and Ngn3 in pancreatic islet.The LSD was used to analyse the data if they fitted normal distribution and homogeneity of variance.Otherwise,non-parametric tests should be used to analyse the data.Dunnett T3 was utilized to analyze the data if they just fitted normal distribution.When P < 0.05 was considered as significant difference.ResultsCompared with that of control group,the FBG increased significanty in model group(P < 0.0001).Compared with that of model group,the FBG was significantly decreased in HRD intervention groups,especially in HHRD group(P < 0.05).In the test of IPGTT and IPITT,compared with control group,the blood glucose in model group was much higher(P < 0.0001).Compared with that of model group,the blood glucose at 90 min and 120 min in LHRD and SAX groups dropped significantly in the test of IPGTT and that of glucose at 60 min,90min and 120 min in SAX group was reduced significantly in the test of IPITT(P < 0.05),while the blood glucose only at 90 min in in the test of IPGTT and 120 min in the test of IPITT in HHRD group was reduced significantly(P < 0.05).In terms of FINS,the levels in model group increased comparing with control group and that of in HHRD and SAX also improved comparing with model group(P < 0.05,P < 0.001).The levels of AUC in IPGTT and HOMA-IR were increased significantly in model group compared with that of control group.Compared with that of model group,both LHRD and HHRD groups amelioated the AUC and HPMA-IR significantly(P < 0.05,P < 0.01).In addition improving the metabolism of glucose,the immunofluorescence images showed that the ratio of glucagon+ cells/insulin+ cells was increased obviously in model group compared to those of the control group(P < 0.01).However,those changes could be revised significantly by LHRD and HHRD(P < 0.05,P < 0.01).At the same time,the ratio of Nkx6.1+ Insulin+ cells was reduced significantly in model group(P < 0.05).The LHRD and HHRD improved those cells levels compared to that of model group(P < 0.05).In addition,the Pdx1 levels in islet was diminished obviously while the co-expression of insulin,glucagon and Pdx1 was increased in model group(P < 0.05).Compared with that of model group,the Pdx1 expression increased while the coexpression of insulin,glucagon and Pdx1 was decreased significantly in HHRD group(P <0.05).Compared with that of control group,the level of Caspase 12 in islet was increased significantly in model group.After intervention,the expression of Caspase12 was reduced greatly.Interestingly,the Cleaved Notch1 level was increased and the Ngn3 level in islet was decreased significantly in HHRD group which were changed in model group compared to that of model group(P < 0.05).However,little difference was found in the number,area and morphology of islet,and the expression of Ki67,Bax/ Bcl2,Cleaved caspase 3/ Caspase 3 in the pancreas among groups.ConclusionThe HRD shows dose-dependent effects on glucose metabolism improvement through maintaining pancreatic cells identity which may involve with Notch1-Ngn3 signaling pathway in db/db mice.Section 2 The machanism of bio-active components in Huanglian-RenshenDecoction on revising the pancreatic cell dedifferentiation via the Notch1-Ngn3 signaling pathway in MIN6 cellsObjectionThe dedifferentiation of pancreatic cells is the new insight for T2 DM.To detect the machanism of bio-active components berberine(BBR)and ginsenoside Rb1(Rb1)in Huanglian-Renshen-Decoction on revising the pancreatic cell dedifferentiation via Notch1-Ngn3 signaling pathway in MIN6 cells.MethodMTT was used to detect MIN6 cells viability after treatment with different interventions including palmitate(PA),Rb1,BBR and Notch1 specific inhibitor DAPT.Based on the cells viablity,we examined the protein and m RNA levels of Nkx6.1,Ngn3 and Caspase3 in futher to find out the appropriate concentrations of PA.According to the different intervention,we set up control group,PA group,Rb1 group,BBR group,RB group(Rb1+BBR)group and RBD group(Rb1+BBR +DAPT).Elisa kit was used to detect the insulin level after glucose stimulation insulin screation test(GSIS).The glucose expenditure in cell supernatant was detected by Glucose Oxidase method.Immunofluorescence showed the levels of Nkx6.1,Ki67,Ngn3 and Cleaved Notch1.Western Blotting technique was used to determine the protein levels of Nkx6.1,Ngn3 and Cleaved Notch1,Cleaved caspase3/Caspase3 and Caspase12 in cells after treatment.Rt PCR method was applied to determine the m RNA levels of Nkx6.1,Ngn3.The LSD was used to analyse the data if they fitted normal distribution and homogeneity of variance.Otherwise,non-parametric tests should be used to analyse the data.Dunnett T3 was utilized to analyze the data if they just fitted normal distribution.When P < 0.05 was considered as significant difference.Results0.1m M and 0.2m M PA decreased MIN6 cells viability(P < 0.01,P< 0.0001).Those two concentrations of PA were used to intervine the cells with 0h,2h,4h,8h,16 h and 24 h.Compared with that of control group,the expression of Nkx6.1 was decreased while the Ngn3 m RNA was increased significantly after 24 h intervention(P < 0.05,P < 0.01,P< 0.0001).The levels of caspase 3 had little change.Those data indicated that 0.2m M PA with 24 h was suitable to set up the dedifferentiating model.According to the results of MTT,the concentrations of Rb1,BBR and DAPT with 40μmol/L,2.5μmol/L and 20μmol/L,respectively were appropriate.Compared with that of control group,the protein and m RNA levels of Nkx6.1 in model group were decreased significantly(P < 0.01,P< 0.001).While,the m RNA transcription and protein translation levels of Nkx6.1 increased in Rb1 and RB groups(P < 0.05).The similar tendency was shown in immunofluorescence images.The apoptosis proteins of Caspase12 and Cleaved caspase3/Caspase3 detected with Western Blotting and proliferation marker Ki67 tested with immunofluorescence in any groups showed no significant difference.The immunofluorescence images showed that Cleaved Notch1 in model group was decreased significantly while the Rb1,BBR and RB could ameliorate this.However,the RBD revesed the function of RB.Compared with that of control group,the m RNA levels of Ngn3 went up in model group(P < 0.01).Coampared with that of model group,Rb1,BBR and RB reduced the m RNA expression while the levels in RBD group ascended(P < 0.01,P < 0.05).Western Blotting showed no difference in Ngn3 protein contents in any groups.GSIS results showed that the supernatant insulin concentrations stimulated by 2.8m M glucose in model group was much higher than that of control group.Compared with that of model group,the insulin screation increased in all treatment groups,except BBR group.Under circumstance of 16.7m M glucose,the insulin levels in model group decreased while the levels in all treatment groups increased significantly,especially in BBR group.The cell glucose comsuption in model group decreased significantly compared with that of control group(P < 0.01).The BBR and RB could increased cell glucose comsuption(P < 0.01,P < 0.05).Conclusion0.2mmol/L PA can induce the dedifferentiation of MIN6 cell after 24 h.The bio-active components in Huanglian-Renshen-Decoction improve the cell dedifferentiation,especially Rb1 via regulating Notch1-Ngn3 signaling pathway.