| Objective:To investigate the effects of c SN50.1 on the expression of EMT-related factors α-SMA,Vimentin and E-cadherin in HK-2 cells induced by high glucose and the role of c SN50.1 in NF-κB signaling pathway,the effect of c SN50.1 on the transdifferentiation of HK-2 cells was investigated.Methods:1.The effects of different concentrations of c SN50.1(0μM,10μM,30μM,50μM)on the proliferation of HK-2 cells were investigated by CCK-8 assay.2.HK-2 cells were divided into 6 groups :Control group(normal group),HO group(hypertonic group),HG group(high glucose group)and c SN50.1 groups(10μM,30μM,50μM).Morphological changes of cells in each group were observed under inverted microscope.3.The q RT-PCR and Western Blot were used to detect the expression of α-SMA,Vimentin and E-cadherin in each group.4.HK-2 cells were divided into 5 groups :Control group,HO group,HG group,PDTC group(NF-κB inhibitor)and c SN50.1 group.5.The expression of NF-κB protein in cytoplasm and nucleus of HK-2 cells was detected.Results:1.The results of different concentrations of c SN50.1 on HK-2 cell proliferation showed that compared with 0 μM group,the absorbance value of 10 μM group and 30 μM group had no significant change(P > 0.05),while the absorbance value of 50 μM group was significantly decreased(P < 0.01).2.The results of c SN50.1 on the morphological changes of HK-2 cells induced by high glucose showed that the cells in the Control group showed a "paving stone" shape,typical epithelioid cell morphology.There was no significant change in HO group compared with Control group.The cells in HG group were long spindle shaped with different sizes and shapes.After c SN50.1 intervention,the long spindle cells in 10 μM group,30 μM group and 50 μM group were significantly reduced,but the number of cells in 50 μM group was decreased.3.The effects of c SN50.1 on the expression of EMT-related factors α-SMA,Vimentin and E-cadherin in HK-2 cells induced by high glucose showed that:(1)q RT-PCR results showed:(1)α-SMA m RNA expression:Compared with the Control group,the expression of HO group had no significant change(P > 0.05),the expression of HG group,10 μM group and30 μM group was significantly increased(P < 0.01),and the expression of 50 μM group was significantly increased(P < 0.05).Compared with HG group,the expression in 10 μM group had no significant change(P > 0.05),and was significantly decreased in 30 μM and50 μM groups(P < 0.01).(2)Vimentin m RNA expression:Compared with the Control group,the expression of HO group had no significant change(P > 0.05),but the expression of HG group was significantly increased(P < 0.01),10 μM group,30 μM group and 50 μM group were significantly increased(P < 0.05);Compared with HG group,the expression in 10 μM group had no significant change(P > 0.05),and was significantly decreased in 30 μM and50 μM groups(P < 0.05).(3)E-cadherin m RNA expression:Compared with the Control group,the expression of HO group had no significant change(P > 0.05),HG group significantly decreased(P <0.01),10 μM group,30 μM group and 50 μM group significantly decreased(P <0.05);Compared with HG group,10 μM group,30 μM group and 50 μM group were significantly higher(P < 0.01).(2)The results of Western Blot experiment showed:(1)α-SMA protein expression:Compared with the Control group,the expression of HO group had no significant change(P > 0.05),the expression of HG group was significantly increased(P < 0.01),the expression of 10 μM group,30 μM group and 50 μM group was significantly increased(P < 0.05);Compared with HG group,the expression of 10 μM group had no significant change(P > 0.05),30 μM group significantly decreased(P <0.01),50 μM group significantly decreased(P < 0.01).(2)Vimentin protein expression:Compared with the Control group,the expression of HO group had no significant change(P > 0.05),but it was significantly increased in HG group,10 μM group,30 μM group and 50 μM group(P < 0.05).Compared with HG group,the expression in 10 μM group had no significant change(P > 0.05),and was significantly decreased in 30 μM and 50 μM groups(P < 0.05).(3)E-cadherin protein expression:Compared with the Control group,the expression of HO group had no significant change(P > 0.05),HG group and 10 μM group significantly decreased(P < 0.01),30 μM group and 50 μM group significantly decreased(P <0.05);Compared with HG group,10 μM group,30 μM group and 50 μM group were significantly higher(P < 0.01).4.Effects of c SN50.1 on high glucose-induced HK-2 cells mediated by NF-κB signaling pathway:(1)Effects of NF-κB signaling pathway on HK-2 cells induced by high glucose in c SN50.1 :(1)α-SMA protein expression:Compared with the Control group,the expression of HO group had no significant change(P > 0.05),the expression of HG group and c SN50.1group was significantly increased(P < 0.01),and PDTC group was significantly decreased(P < 0.05).Compared with HG group,the expression of PDTC group and c SN50.1 group were significantly decreased(P < 0.01).(2)Vimentin protein expression:Compared with the results of Control group,the expression of HO group had no significant change(P > 0.05),the expression of HG group and c SN50.1 group were significantly increased(P < 0.01),PDTC group was significantly increased(P < 0.05);Compared with HG group,the expression of PDTC group was significantly decreased(P < 0.01),and c SN50.1 group was significantly decreased(P <0.05).(3)E-cadherin protein expression:Compared with the Control group,the expression of HO group had no significant change(P > 0.05),and the expression of HG group,PDTC group and c SN50.1 group were significantly decreased(P < 0.01).Compared with HG group,the expression of PDTC group was significantly higher(P < 0.01),and c SN50.1group was significantly higher(P < 0.05).(2)NF-κB protein expression:(1)The expression of NF-κB protein in the cytoplasm: Compared with the Control group,the expression of HG group and c SN50.1 group was significantly increased(P <0.01),while the expression of HO group and PDTC group had no significant change(P >0.05).Compared with HG group,the expression of PDTC group was significantly decreased(P < 0.01),while c SN50.1 group had no significant change(P > 0.05).(2)The expression of NF-κB protein in the nucleus:Compared with the Control group,the expression of NF-κB protein in HO group had no significant change(P > 0.05),but was significantly increased in HG group(P < 0.01),PDTC group and c SN50.1 group(P <0.05).Compared with HG group,the expression of PDTC group was significantly decreased(P < 0.01),while that of c SN50.1 group was significantly decreased(P < 0.05).Conclusion:1.cSN50.1 could improve the transdifferentiation of HK-2 cells induced by high glucose.2.The effect of c SN50.1 on the transdifferentiation of HK-2 cells induced by high glucose may be achieved by decreasing the nuclear NF-κB transport and thereby affecting the expression of α-SMA,Vimentin and E-cadherin. |
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