| Objective:In this study,collecting duct cells?IMCD3 cell line?as the study object,to observe the changes of apoptosis and AQP2 protein expression induced by doxorubicin induced IMCD3 cells injury;further study of Danggui Shaoyao San on the induction of IMCD3 after adriamycin induced cAMP,PKA Changes in the expression of AQP2,Na+-K+-ATPase protein,and preliminary study on the mechanism of Danggui Shaoyao San in the injury of IMCD3 cells induced by adriamycin..Methods:Part one.Study on induced injury of IMCD3 cells with different concentrations of adriamycin.The MTT assay was used to detect the injury of IMCD3 cells with different concentrations of adriamycin.Observed the effect of different concentrations of adriamycin,IMCD3 cells growth state.2.Inverted microscope observation,with the change of adriamycin concentration,IMCD3 cell morphology changes.3.Annexin V/PI staining was used to observe the gradient changes of adriamycin and observed the apoptosis of IMCD3 cells.Western blot was used to detect the effect of AQP2 protein expression in IMCD3 cells with adriamycin concentration.The MTT assay and Annexin V/PI staining were divided into normal group,1×10-7mol/L group,1×10-88 mol/group,1×10-99 mol/L group,and 1×10-1010 mol/L group,1×10-11mol/L group;Western blot was divided into normal group,1×10-88 mol/group,1×10-9mol/L group,1×10-1010 mol/L group,1×10-11mol/L group.Part two:Effects of Danggui Shaoyao Sanr with Different Concentrations on the Expression of AQP2,Na+-K+-ATPase Protein in IMCD3 Cells induced by Adriamycin.1.MTT assay was used to detect the effect of Danggui Shaoyao San on the growth of IMCD3 cells damaged by Adriamycin.Western blot was used to detect the expression of AQP2,Na+-K+-ATPase protein in IMCD3 cells induced by Adriamycin with different concentrations of Danggui Shaoyao San.3.MTT assay was divided into normal group,model groupp,0.2mg/mL,0.4mg/mL,0.6mg/mL,0.8mg/mL,1.6mg/mL,3.2mg/mL,6.8mg/mL,12.8mg/mL;Westernblotassay were divided into normal group and model group,0.2mg/ml,0.4mg/mL,0.6mg/mL,0.8mg/mL,1.6mg/mL,3.2mg/mL.Part three:Mechanism of Adriamycin-induced IMCD3 Cell treated by Danggui Shaoyao San.1.The changes of cAMP content in IMCD3 cells induced by adriamycin were detected by ELISA.2.Detection of Na+-K+-ATPase Activity in IMCD3 Cells Induced by Adriamycin in Na+-K+-ATPase Assay Box3.The expression ofPKA,CREB,AQP2 and Na+-K+-ATPase protein in IMCD3 cells induced bydetected were detected by Western blot,and the mechanism of Danggui Shaoyao San on adriamycin induced IMCD3 cell injury was detected.The experiments were divided into normal group,model group,low dose group of Danggui Shaoyao San,middle dose group of Danggui Shaoyao San,high dose group of Danggui Shaoyao San and H-89 inhibitor group.Results:Part one.Effects of AQP2 protein expression in IMCD3 cells in different concentrations of adriamycin.1.The results of MTT assay on IMCD3 cell injury with different concentrations of doxorubicin showed that compared with the normal group,the adriamycin concentration was more than 1×10-77 mol/L,the cells basically died and the OD value decreased,showing that the cells showed different degrees of the inhibitory effect was significantly different?P<0.01?;when the concentration was reduced to 1×10-99 mol/L,1×10-1010 mol/L,and 1×10-1111 mol/L,the OD value increased,and the difference was significant?P<0.01?.2.The results of Annexin V/PI staining showed that compared with the normal group,the apoptosis rate increased gradually when the adriamycin concentration was 1×10-8mol/L,1×10-99 mol/L,1×10-10mol/L,1×10-1111 mol/L,and the difference was significant?P<0.01?.3.The results of Western blot showed that,compared with the normal group,the concentration of adriamycin was 1×10-88 mol/L can significantly decrease the expression of AQP2?P<0.01?;Adriamycin concentration was 1×10-10,1×10-1111 mol/L,AQP2protein expression increased at different degree?P<0.01?.The results of Annexin V/PI staining showed that compared with the normal group,the apoptosis rate of adriamycin was increased at 1×10-8mol/L,1×10-9mol/L,1×10-10mol/L,1×10-11mol/L,and the difference was significant?P<0.01?.The results of comprehensive MTT,Annexin V/PI staining and Western blot method were applied.The doxorubicin concentration of1x10-8mol/L was used as the concentration of induced injury IMCD3 as the best concentration for subsequent experiments.Part two:Effects of Danggui Shaoyao San with Different Concentrations on the expression of AQP2,Na+-K+-ATPase Protein in IMCD3 Cells induced by Adriamycin.1.MTT assay with different concentrations of Danggui Shaoyao San on Adriamycin-induced IMCD3 cells showed that compared with the normal group,Danggui Shaoyao Powder was 0.2mg/ml,0.4mg/ml,0.6mg/ml,0.8mg/ml,1.6mg There was no significant effect on the growth and proliferation of normal IMCD3 cells at the concentrations of ml and 3.2 mg/ml?P>0.05?,and 6.4 mg/ml and 12.8 mg/ml inhibited the growth and proliferation of IMCD3 cells to different degrees?P<0.05?.Compared with the model group,0.2mg/ml,0.4mg/ml,0.6mg/ml,0.8mg/ml,1.6mg/ml,3.2mg/ml had no significant effect on the growth and proliferation of IMCD3 cells?P>0.05?.6.4 mg/ml and 12.8mg/ml had different degrees of inhibition on the growth and proliferation of IMCD3cells and the difference was statistically significant.2.The results of Western blot showed that the expression of AQP2 and Na+-K+-ATPase in the model group was significantly lower than that in the normal group?P<0.01?.Compared with the model group,AQP2,Na+-were increased with the increase of the concentration of Danggui Shaoyao San.The expression of K+-ATPase protein was significantly increased and showed a concentration-dependent manner,with the most significant increase at 1.6mg/ml?p<0.01?.Based on the results of MTT assay and Western blot assay,0.2mg/ml,1.6mg/ml,and 3.2mg/ml were used as experimental groups for the low,middle,and high doses of Danggui Shaoyao San in the later experiments.Part one.Effects of AQP2 protein expression in IMCD3 cells in different concentrations of adriamycin.1.The results of Na+-K+-ATPase activities assay showed that Na+-K+-ATPase activities in the model group were significantly lower than that in the normal group?P<0.01?.Compared with the model group,Danggui Shaoyao san could increase Na+-K+-ATPase activities in each dose group.in which the increase in Na+-K+-ATPase activity is most pronounced in the medium dose.There was no significant difference in Na+-K+-ATPase between the H-89 inhibitor group and the model group.2.ELISA assay of danggui Shaoyao san showed that the cAMP content of IMCD3 cells induced by adriamycin was significantly lower in the model group than in the blank control group?P<0.05?.Compared with the model group,the doses of cAMP in the Danggui Shaoyao san group had With different degrees of improvement,the middle dose of Danggui Shaoyao san was most obvious,and the cAMP levels in different H-89inhibitor groups were significantly lower than those in the model group?P<0.05?.3.The expression of PKA,CREB,AQP2,and Na+-K+-ATPase in IMCD3 cells induced by adriamycin were detected by Western blot assay.The results showed that compared with the blank control group,PKA,CREB,AQP2,and Na+-K+ATPase in the model group were significantly decreased?P<0.01?.Compared with the model group,the expressions of PKA,CREB,AQP2,and Na+-K+-ATPase were increased in dose-dependent manner in each dose group of Danggui Shaoyao san.Compared with the model group,H-89inhibitor group had significantly lower PKA,CREB,and AQP2?P<0.05?,and there was no significant difference in Na+-K+-ATPase protein expression.Conclusions:1.Adriamycin can induce apoptosis of IMCD3 cells and inhibitor the expression of AQP2 protein.2.Danggui Shaoyao san can improve the expression of AQP2 and Na+-K+-ATP enzyme protein in IMCD3 cells induced by Adriamycin.Danggui Shaoyao san can improve the expression of AQP2 and Na+-K+-ATP enzyme in IMCD3 cells.The enhancement of cell water balance regulation may be achieved by activating the cAMP/PKA signal transduction pathway. |
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