ABSTRACT
To explore a new underlying molecular mechanism of Huangkui Extract Powder (HKEP) in the alleviation of diabetic nephropathy (DN). Murine immortalized podocytes were divided into (i) normal glucose (NG, 5.6 mM), (ii) NG + HKEP (0.45 g/L), (iii) HG, and (iv) HG + HKEP (0.45 g/L) groups. MTT assay and flow cytometry were used to detect the podocyte proliferation, apoptosis and cell cycle. Cell viability was inhibited, and apoptosis increased in(iii) HG group compared with (i) NG group (p<0.05). mRNA and protein expression of nephrin and podocin significantly decreased in (iii) HG group compared with (i) NG group (p<0.05). When compared with (iii) HG group, (iv) HG + HKEP group had higher cell viability, lower apoptotic rate and higher mRNA and protein expression of nephrin and podocin (p<0.05). HKEP can attenuate HG-induced podocyte damage, which may be one of the mechanisms of HKEP for attenuating DN.
Explorar un nuevo mecanismo molecular subyacente del extracto del polvo de Huangkui (HKEP) en el alivio de la nefropatía diabética (ND). Los podocitos murinos inmortalizados se dividieron en (i) grupos de glucosa normal (NG, 5,6 mM), (ii) NG + HKEP (0,45 g/L), (iii) HG y (iv) HG + HKEP (0,45 g/L). Se utilizaron el ensayo MTT y la citometría de flujo para detectar la proliferación de podocitos, la apoptosis y el ciclo celular. La viabilidad celular se inhibió y la apoptosis aumentó en el grupo (iii) HG en comparación con el grupo (i) NG (p<0,05). La expresión de ARNm y proteínas de nefrina y podocina disminuyó significativamente en el grupo (iii) HG en comparación con el grupo (i) NG (p<0,05). En comparación con el grupo (iii) HG, el grupo (iv) HG + HKEP tuvo una mayor viabilidad celular, una tasa de apoptosis más baja y una expresión de ARNm y proteínas más altas de nefrina y podocina (p<0,05). HKEP puede atenuar el daño de los podocitos inducido por HG, que puede ser uno de los mecanismos de HKEP para atenuar la DN.
Subject(s)
Plant Extracts/administration & dosage , Diabetic Nephropathies/drug therapy , Podocytes/drug effects , Powders , Plant Extracts/genetics , Cell Cycle , Blotting, Western , Apoptosis/drug effects , Cell Culture Techniques , Reverse Transcriptase Polymerase Chain Reaction , GlucoseABSTRACT
Objective To investigate the effect of Huangkui extract powder (HK) on the expression of nephrin and podocin proteins in mouse podocytes induced by high glucose,which is involved in the treatment of diabetic nephropathy (DN).Methods Cultured mouse podocytes (MPC5) were incubated in high glucose and HK at 5.6 mmol/L NG,5.6 mmol/L NG + 0.45 g/L HK,25 mmol/L HG,25 mmol/L HG + 0.45 g/L HK,respectively.The 5.6 mmol/L NG group was used as normal control.After 24 hours of intervention,we detected podocyte apoptosis by Annexin-V FITC/PI double staining,measured the mRNA and protein expression of nephrin and podocin by qRT-PCR and Western blot.Results Compared with the control group (5.6 mmol/L,NG),the apoptosis rate of podocytes in the high glucose concentration group (25 mmol/L,HG) was significantly higher [(20.39 ± 0.03) ％ vs.(17.70 ± 0.91) ％,t =2.947,P ＜ 0.05)].The apoptosis rate of podocytes in the 25 mmol/L HG + 0.45 g/L HK group was significantly lower than that in the 25 mmol/L HG group [(11.96 ± 1.11) ％ vs.(20.39 ± 0.03) ％,t =7.586,P ＜ 0.01].The results of qRT-PCR and Western blot showed that the expression of nephrin and podocin was significantly inhibited by high glucose concentration compared with the control group[(0.489 ±0.040) vs.(0.721 ±0.022),t =4.992,P ＜0.01;(0.387 ±0.014) vs.(0.778 ±0.036),t =10.050,P ＜0.01],and the expression of podocin and nephrin was increased by appropriate concentration of H K [(0.603 ± 0.013) vs.(0.489 ± 0.040),t =2.653,P＜0.05;(0.640±0.024) vs.(0.387 ±0.014),t=8.946,P＜0.01].Conclusion Podocyte apoptosis can be induced by prolonged high glucose treatment,but a certain concentration of HK can inhibit podocyte death induced by high glucose.The possible mechanism is that HK may inhibit the apoptosis of podocytes by regulating the expression of podocin and nephrin in podocytes at high glucose concentration,thus plays a protective role on podocytes.