The Effects Of Baicalin Bai On HK-2 Cells Apoptosis Induced In High Glucose Condition

ObjectiveTo study the apoptosis effect of Baicalin Bai(BB)on HK-2 cells by high glucose.To provide experimental basis for curative effect on DN with Chine se medicine.Method1.Filtrated the growth curve of HK-2 cells and the drug concentration of Baicalin Bai and Rosiglitazone:Cultured HK-2 cells,and detected 1000 ce 11s/well,2000 cells/well,4000 cells/well and 8000 cells/well by M TT assay at 24h,48h,72h,96h and 120h.Then fit the growth trend curve to screen the cell density.The proliferation of HK-2 cells was detected by M TT assay at a cell density of 4000 cells/well at 24 h,48 h,72 h,and th e drug concentration was determined by calculating the inhibitory rate.2.Cell proliferation test:Cultured HK-2 cells,and(50 μmol/L,100μmol/L,200μmol/L)for different concentrations of the positive contr ol drug(5μmol/L,10μmol/L)under high glucose for 24 hours,48 hours and 72 hours respectively by MTT assay of HK-2 cell proliferation.3.Cell apoptosis detection.Detection the morphology of cell apoptosis by hoechest33342 staining and Annexin V-FITC/PI staining method was used for the quantitative determination of cell apoptosis.4.cell apoptosis antibody test:detection how Baicalin Bai influenced the expression of cell apoptosis related Bax、Bcl-2、PKC、TGF-β by immunohis tochemistry(IHC).Result1.Filtrated the growth curve of HK-2 cells and the drug concentration of Baicalin Bai and Rosiglitazone.(1)Filtrated the growth curve of HK-2 cells:By measuring the growth cu rve of HK-2,it was found that the cell density of 4000/well showed an ex ponential growth trend with the prolongation of incubation time,which acco rds with the normal growth cycle.Select a cell density of 4000/well for subsequent experiments.According to the proliferative phase of the cells,the time points of 1,3 to 1/2 of the exponential proliferative phase were selected as 24h,48h and 72h.(2)Filtrated the drug concentration of Rosiglitazone:The 0D value of lo w glucose group,high glucose group and rosiglitazone group(4 groups)was significantly lower than that of control group,Respectively,from 0.399±0.061 at 24h,0.874 ±0.073 at 72h,0.301 ±0.026 at 24h,0.561 ± 0.024 at 72h,0.368 ± 0.043 at 24h,0.743 ± 0.021 at 72h,0.395 ± 0.013 at 24h,0.707 ± 0.048 at 72h,0.366 ± 0.018 at 24 h,0.686 ± 0.011 at 72 h,0.329 ± 0.039 at 24 h,and 0.392 ± 0.020 at 72 h.When the concentration of rosiglitazone is over 20 μmol/L,the toxicity of HK-2 cells is high,s o the concentration of 5μmol/L and 10 μmol/L is the positive control d rug concentration.(3)Filtrated the drug concentration of Baicalin Bai:The 0D value of low glucose group,high glucose group and Baicalin Bai group(5 groups)was si gnificantly lower than that of control group,Respectively,from 0.441±0.014 at 24h,0.788±0.028 at 72h,0.494±0.021 at 24h,0.618±0.024 at 72h,0.462±0.014 at 24h,0.575±0.021 at 72h,0.517±0.029 at 24h,0.544±0.040 at 72h,0.506±0.007 at 24h,0.608±0.038 at 72h,0.592±0.059 at 24h,0.561 ± 0.027 at 72h and 0.343 ± 0.038 at 24h,0.221 ± 0.025 at 72h.When the con centration of Baicalin Bai is over 200 μmol/L,the toxicity of HK-2 cells is high,so the concentration of 50μmol/L,100 μmol/L and 200 μmol/L is t he positive control drug concentration.2.MTT assay:The 0D values of low glucose group,high glucose group,r osiglitazone group and baicalin group were detected by MTT assay at 24h,48 h and 72h.The 0D of the three groups was 0.331 0.017 to 72h 0.384 0.014 to 24 h 0.742 0.031 24 h 0.419 0.024 to 72 h 0.734 0.079 24 h 0.382 0.035 to 72 h 0.646 0.039 24 h 0.512 0.019&It;RTI ID 二 0.0>0.857 ± 0.046 a t 72 h,0.535 ± 0.011 at 24 h,0.789 ± 0.042 at 24 h,0.492 ± 0.041 at 24 h and 0.562 ± 0.101 at 72 h.The results showed that baicalin could promo te the proliferation of HK-2 cells stimulated by high glucose.3.Cell apoptosis detection:Apoptosis was detected by AnnexinV-FITC/P I double staining,low-sugar group,rosiglitazone group(two groups)apopto sis rate with time prolonged slight upward trend,respectively,from the 24 h of 2.97 ± 0.91%to 72h of 3.73 ± 2.19%,24h of 6.73 ± 3.25%to 72h of 7.03 ± 2.57%,24h of 5.20 ± 0.20%to 72h of 6.27 ± 2.83%;high glucose g roup,baicalin group(three groups)of apoptosis rate with time decreased f rom 7.70± 4.76%,respectively,of 24h to 72h of 7.27 ± 3.15%,from 7.57 ± 3.07%of 24h to 72h of 3.60 ± 1.40%,from 24h of 6.33 ± 1.82%to 72h o f 5.53 ± 2.45%,from 7.83 ± 4.57%of 24h to 72h of 4.93 ± 3.11%.The resu Its showed that Baicalin could inhibit high glucose-induced tubular epithel ial cell apoptosis,apoptosis rate was concentration-dependent manner.4.Detection of apoptosis factor test:By immunohistochemistry,we obser ved that Bax,Bcl-2,TGF-β,the expression of PKC in the low glucose group was negative;wherein Bax,TGF-β1,PKC in high glucose group was positive,rosiglitazone group,baicalin group was weakly positive,as the concentrat ion increases,the positive is not obvious.The expression of Bcl-2 was posi tive in the high glucose group.The positive rate of Bcl-2 expression was s trong in the rosiglitazone group and baicalin group,and increased with the increase of the concentration.Baicalin reduced Bax/Bcl-2 ratio may be r educed apoptosis by regulating the expression of TGF-β family.ConclusionBaicalin Bai could reduce HK-2 cell apoptosis and promote cell prolifer ation by high glucose.