| ObjectiveThe mechanism of Niaoduling and its active components in the treatment of renal fibrosis and diabetic nephropathy was discussed through the regulation of the TGF-β1/Smads signaling pathway of renal tubular epithelial cells(HK-2)and its effect on the morphology,growth cycle and migration ability of HK-2 cells through the Niaoduling and its active ingredient,Icariin-I and Ginsenoside Rd.Methods1.Establishment of cell model:the doge of 2ng/mL TGF-β1 induced HK-2 cells;According to the mode of administration,they are divided into GrA,Gr-B,Gr-C,Gr-D;Gr-A:giving drugs for 24h firstly,then adding 2ng/mL TGFβ1 for 30min;Gr-B:giving drugs for 24h firstly,then adding 2ng/mL TGF-β1 for 24h;Gr-C:giving experimental drugs and 2ng/mL TGF-β1 at the same time for 30min;Gr-D:experimental drugs and 2ng/ml TGF-β1 at the same time for 24h;The drugs in each experimental group were Niaoduling,Ginsenoside Rd and Icariin-I.2.Screening of the concentrations of ginsenoside Rd and Icariin-I:The effect of two monomers on the proliferation of HK-2 cells was determined by MTT.3.Changes of cell migration ability in HK-2:the effect of drug on the migration ability of HK-2 cells in Gr-B was determined by scratch experiment.4.Study on the morphology and growth cycle of HK-2 cells:The changes of cell morphology and the changes of growth cycle before and after cell treatment were observed by automatic inverted microscope and flow cytometry.5.Determination of gene expression of HK-2 cells:The gene expressions of Smad2,Smad3,Smad4,Smad7 and Smurf2 in two ways of Gr-A and Gr-B were determined by PCR.6.Determination of HK-2 cell protein expressions:The expressions of Smad4 protein,Smad2,Smad3 protein and phosphorylation in two modes of administration of Gr-A and Gr-C were detected by western blot experiment.The expression of Smad7 protein in two modes of administration of Gr-B and Gr-D were detected by western blot experiment.Results1.The safe concentration range of the drug was determined by MTT,and the working concentration of three drugs was finally determined:IcariinI:1μg/ml,5 μ g/mL,25 μ g/mL;Ginsenoside Rd:50 μ g/ml,100 μ g/mL;Niaoduling:100 μ g/mL,500 μ g/mL,1000 μ g/mL.2.Morphological changes of HK-2 cells:blank group cells is oval,that is"paving stone" sample form.In the Gr-A group,the model group was not different from the blank group,and it was still a paving stone sample,and in the Gr-B group,the model group cells became longer,manifested as fibroblast-like morphology,and the morphological fiber samples of the Icariin-I group and the Niaoduling group cells decreased and gradually approached the normal cells,and the role of Ginsenoside Rd was not obvious.3.The results of migration experiment showed that cell fusion efficiency of the model group was significantly increased compared with the blank group in Gr-B,the middle dose group of Icariin-I,ginsenoside Rd in 50 μ g/mL,and the high dose group of Niaoduling could significantly inhibit cell migration.4.The results of HK-2 cell cycle showed that Gr-A:the model group and each dosing group had little effect on the HK-2 cell cycle,Gr-B:compared with the blank group,the model group G2/M period’s cells had obvious decrease(P<0.05),Icariin-I,Ginsenoside Rd and Niaoduling poison group had effects on it’s period.S period:the model group decreased slightly,Icariin-I and Ginsenoside Rd did not increase,and the high dose group of Niaoduling could significantly improve this trend(P<0.05),so Niaoduling had the greatest effect on cell cycle in the role of drugs.5.The results of HK-2 cell gene expression showed that Gr-A:compared with the blank group:The difference of gene expression between Smad2,Smad3,Smad4,Smad7 and Smurf2 was not statistically significant(P>0.05).Compared with the model group,Smad2,Smad3 and Smad4:There was no obvious effect in each drug delivery group.Smad7:Icariin-I,Ginsenoside Rd and Niaoduling can promote gene expression,among which,Icariin-I high dose group,ginsenoside Rd High dose group significantly increased its gene expression(P<0.05);The contribution of Niaoduling concentration increased the expression of proteins(P<0.01);Smurf2:Ginsenoside Rd also has a good role in promoting,especially in the high dose group(P<0.01),while Niaoduling has a tendency to reduce its expression.Gr-B:compared with the blank group,the gene expression of Smad2 and Smad4 showed no significant change,the gene expression of Smad3 decreased significantly(P<0.001),the gene expression of Smad7 and Smurf2 increased significantly(P<0.45).6.Western blot method measured,Gr-C:compared with the blank group,pSmad2,Smad2,p-Smad3,Smad3:significantly increased(P<0.05,P<0.05,P<0.01,P<0.05).Compared with the model group,the high dose of Niaoduling reduced the expression of p-Smad2,p-Smad3 and Smad3 protein(P<0.05)and the Icariin-I and the ginsenoside Rd had no obvious effect on the expression of these proteins.Gr-A:compared with the blank group,the expression of p-Smad2、Smad2、p-Smad3 and Smad3 protein increased significantly,the difference was statistically significant(P<0.05、P<0.05、P<0.01、P<0.05),Smad4:there was an upward trend,but the difference was not statistically significant.Compared with the model group,p-Smad2:the high dose group of ginsenoside Rd significantly reduced,the difference was statistically significant(P<0.05),and the difference of high dose Niaoduling group was statistically significant(P<0.05).p-Smad3,Smad3 and Smad4:Icariin-I and Ginsenoside Rd had no effect;the high dose group of Niaoduling had a downward effect,and the difference was statistically significant(P<0.05).Gr-B、Gr-D:compared with the blank group,the expression of Smad7 protein increased slightly,but the difference was not statistically significant,but the drug delivery group has no effect.Conclusions1.2ng/mL TGF-β1 induced HK-2 cells for 24h,the morphology of HK-2 cells is from paving stones to fibroblasts,and cell migration ability was increased,and the drug groups can reduce the change of cell morphology to varying degrees,Inhibits the migration of HK-2 cells induced by TGF-β1,which has a certain protective effect on the kidneys.2.Icariin-I,Ginsenoside Rd and Niaoduling poison group all increased the G2/M stage cells of HK-2 cells,and the number of cells in the S stage was significantly raised by Niaoduling,indicating that the effect of Niaoduling on HK-2 cell cycle was obvious than that of Icariin-I and Ginsenoside Rd.3.In Gr-A way,there was no significant difference in gene expression of index proteins in the model group,but the proteins increased significantly,that is,Smad2 and Smad3 were obviously activated,the phosphorylation level of Smad2 and Smad3 increased,the Smads signaling pathway was activated,and the protein expression of Smad3 was greatly improved.In this process,the morphology,growth cycle and gene expression of HK-2 cells were not affected.The drug group had a certain effect on the gene and protein expression of HK-2 cells,and the Niaoduling was significantly more effective than Icariin-I and Ginsenoside Rd.4.In Gr-B way:the expression of Smad3 gene in the model group was significantly reduced,and the gene expression of Smad7 and Smurf2 increased significantly,when the growth cycle and cell morphology of HK-2 cells changed,and the migration ability of cells increased.5.The expression of p-Smad2,Smad2,p-Smad3 and Smad3 protein in the cells of the model group increased significantly,which showed that the TGFβ1 had activated the TGF-β 1/smads signaling pathway,and the Niaoduling had a significant inhibitory effect on it,whether it was first given and then modeled at the same time for 30min.The protein expression of p-Smad2,pSmad3 and Smad3 was significantly reduced.The high dose of ginsenoside Rd can only significantly reduce the expression of p-Smad2 protein.In general,Niaoduling can inhibit the expression of protein in Smad2 and Smad3,and inhibit their phosphorylation,the role of Icariin-I and Ginsenoside Rd are not as good as the effect of Niaoduling.6.To sum up,Niaoduling and its active ingredients can inhibit the TGFβ1/Smads signaling pathway and play a role in the treatment of renal fibrosis. |
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