The Intervention Study Of Niaoduling And Its Effective Components In The Process Of EMT Induced By TGF-?1

Objective:To investigate the effect and protein mechanism of NiaoDuling and its effective components on the prevention and treatment of renal fibrosis via measurement the change of the cell morphology and the mRNA and protein expression of markers during HK-2 cells transdifferentiation.Methods:1.Construction of EMT cell model upon TGF-?1 stimulation in Renal tubular epithelial cells line HK-2.The optimization of EMT conditions by virtue of testing the variation of cell proliferation and morphology.2.Administration and groupingThe cells were divided into two groups as drug prevention and drug treatment group after EMT challenge.Besides,the low,mid and high dose NiaoDuling(32 ? g/mL,160 ? g/mL,800 ? g/mL)and its effective components Baohuoside I(1 p g/mL,5 ? g/mL,25 ? g/mL)and ginsenoside Rd(50?g/mL,100? g/mL,200 ? g/mL)were decided.3.Monitoring the cell morphology change by cell morphological observation.4.Detecting protein expression of a-SMA and Fibronectin using Immunofluorescence,also the detection of EMT marker protein expression.5.Prevention of HK-2 cell transdifferentiation(drug prevention group)Treat the HK-2 with TGF-? 1 and intervention drugs simultaneously.RT-qPCR was adopted to investigate the mRNA expression of the epithelial marker protein E-cadherin and Claudin-1,mesenchymal cell marker protein of Fibronectin,Snail and a-SMA.Western-blot was introduced to detect the protein expression of these proteins.6.Reverse of HK-2 cell transdifferentiation(drug treatment group)Treat the HK-2 72h with prepared drug reagents after 72h intervention by TGF-?1.RT-qPCR was adopted to investigate the mRNA expression of the epithelial marker protein E-cadherin and Claudin-1,mesenchymal cell marker protein of Fibronectin,Snail and a-SMA.Western-blot was introduced to detect the protein expression of these proteins.Results:1.Optimization EMT conditions of HK-2 cells induced by TGF-?1Dosage of 2.5 ng/mL,5 ng/mL and 10 ng/mL.TGF-?1 can inhibit the proliferation of HK-2 cells.Furthermore,5 ng/mL group rendered the comparative inhibitory effect with that of 10 ng/mL group,exhibiting the strongest inhibition effect on the moment of 72h.Following EMT challenging,Cell morphology changed from typical epithelial cells with tight connection into fiber ectomesenchymal cells with increased intercellular space,indicating that EMT has being experienced in HK-2 cell.Therefore,we choose the dosage of 5ng/mL TGF-?1 as the premium EMT induction condition.2.Drug intervention assay on cell proliferationDosage of 5ng/mL TGF-?1 can statistically inhibit the cell proliferation Compared with the control group,which confirmed successful EMT induction(P<0.05).Prevention group:Low concentration of Baohuoside I has a certain synergetic effect with TGF-?1,which exerted stronger inhibition effect for HK-2 cell proliferation.However,high dosage Baohuoside I can significantly inhibit the role of TGF-?1(P<0.05),moreover,the proliferation ability of HK-2 cells was close to normal levels,which illustrated the bidirectional regulation effect to TGF-?1.Ginsenoside Rd with any assay concentration can obviously decreased the effect of TGF-?1,with the corresponding up-regulation of HK-2 proliferation to the normal levels.The same tendency occurred in the Niaoduling groups,with higher cell viability and statistically dosage-dependent prevention(P<0.05).Therapy group:All dosage of selected Baohuoside I and Niaoduling can enhance the cell viability of HK-2 when compared with control group(P<0.05)with the trend of dosage-dependent.To the opposite,ginsenoside Rd with any experimental concentration exerted the inhibitory effect in terms of cell viability.3.Drugs interplay with HK-2 cells in prevention groupsCompared to model groups,Baohuoside I,ginsenoside Rd,and Niaoduling groups all can inhibit EMT process to some extent.Specifically,the inhibition effect of Baohuoside I and Niaoduling groups had extremely correlation with the dosage concentrations.Immunofluorescence results demonstration that ginsenoside Rd and Niaoduling can reduce the expression of a-SMA,while Baohuoside I made the opposite effect with the other groups.But for Fibronectin,the three groups all exerted obvious inhibitory effect.For the marker protein mRNA levels research,Baohuoside I can up-regulate the mRNA transcription of E-cadherin with the dosage-dependent,but still lower than that in the control group.Moreover,ginsenoside Rd can notably down-regulate E-cadherin mRNA transcription,and Niaoduling had very subtitle effect on E-cadherin mRNA transcription;Baohuoside I showed the up-regulated effect on Claudin-1 mRNA transcription,without significant meaning.Ginsenoside Rd can up-regulate Claudin-1 mRNA transcription with the dosage-dependent,the same result can be seen in Niaoduling group.For snail mRNA transcription,all these groups demonstrated significantly down-regulated effect.But for Fibronectin,only ginsenoside Rd and Niaoduling have positive influence.For the protein expression levels,low dosage Baohuoside I had strongest promotion to Claudin-1 and Fibronectin expression(P<0.05),but the middle dosage of ginsenoside Rd and niaoduling had the most premium effect.4.Drugs regulation with HK-2 cells treatment groupsImmunofluorescence results hints that middle and high dosage of ginsenoside Rd and Niaoduling groups both had significantly down-regulation for Fibronectin expression.Although high concentration of Baohuoside I have inhibitory effect to Fibronectin expression,?-SMA expression had elevated tendency.For the marker protein mRNA levels research,Baohuoside I group had distinct influence on the down-regulation for Claudin-1,E-cadherin andFibronectin mRNA transcription.Low and middle dosage ginsenoside Rd can promote the elevation of E-cadherin,Claudin-1 and Fibronectin mRNA transcription,and the middle dosage of ginsenoside Rd had the most obvious effect on Snail mRNA transcription(P<0.05).Niaoduling exhibited the most prominent effect for Claudin-1 and E-cadherin expression.For the protein expression levels,middle dosage of ginsenoside Rd demonstrated perfect function for Claudin-1 enhancement and Fibronectin inhibition(P<0.05).Conclusion:Different dosage of Baohuoside I and ginsenoside Rd had statistically regulation and intervention for HK-2 cells morphology change,related marker mRNA transcription and protein expression during EMT challenging,which can indeed delay and improve renal fibrosis with the similar function as Niaoduding.