Danggui buxue tang inhibited mesangial cell proliferation and extracellular matrix accumulation through GAS5/NF-κB pathway

Figures

• 

  Figure 1. Mouse renal mesangial cells (SV40 MES-13) were treated with 5.6 mM glucose (NG), 30 mM glucose (HG)+DGTXT (0, 5, 10 and 20%) (A) The expressions of GAS5 in SV40 MES-13 cells were detected in NG, 0% HG, 5% HG, 10% HG, 20% HG groups. (B) Protein levels of IKKin NG, 0% HG, 5% HG, 10% HG, 20% HG groups. ** P<0.01, compared with NG group.## P<0.01, compared with HG group.

• 

  Figure 2. DGT participated in the expression of IKK and the activity of NF-κB regulated by HG via GAS5 (A) SV40 MES-13 cells were transfected with si-GAS5 or si-control. Then, SV40 MES-13 cells were divided into normal glucose (NG) group, HG group, HG+DGTXT (10%) group, HG+DGTXT (10%)+si-control group, HG+DGTXT (10%)+si-GAS5 group. The expressions of IKK were detected in NG group, HG group, HG+DGTXT (10%) group, HG+DGTXT (10%)+si-control group, HG+DGTXT (10%)+si-GAS5 group. (B) The luciferase activity of IKK was measured in NG group, HG group, HG+DGTXT (10%) group, HG+DGTXT (10%)+si-control group, HG+DGTXT (10%)+si-GAS5 group. ** P<0.01, compared with NG group. ##P<0.01, compared with HG group. &P<0.01, compared with HG+ DGTXT+si-control group.

• 

  Figure 3. The interaction between GAS5 and IKK in SV40 MES-13 cells (A) RNA-pull down assay using in vitro-synthesized biotinylated GAS5. Precipitation reactions were conducted using streptavidin beads and then subjected to Western blotting. (B) Protein lysates were prepared and immunoprecipitated with IKK antibody or IgG. The RNA levels of GAS5 in immunoprecipitates were measured by real-time PCR. ** P<0.01, compared with IgG.

• 

  Figure 4. GAS5 regulated the expression of IKK and the activity of NF-κB (A) SV40 MES-13 cells were transfected with pcDNA or pcDNA-GAS5. Then, SV40 MES-13 cells were divided into NG group, HG group, HG+pcDNA group, HG+pcDNA-GAS5 group. The expression of IKK and the luciferase activity of NF-κB were detected in NG group, HG group, HG+pcDNA group, HG+pcDNA-GAS5 group. (B) SV40 MES-13 cells were transfected with si-control or si-GAS5. The expression of IKK and the luciferase activity of NF-κB were detected in si-control and si-GAS5 groups. (C) SV40 MES-13 cells were transfected with si-control or si-GAS5, then treated with Cycloheximide (CHX) for 0, 3, 6 and 9 h. The protein level of IKK was detected in each group. ** P<0.01, compared with NG or si-control. ##P<0.01, compared with HG+pcDNA.

• 

  Figure 5. DGT inhibited the proliferation of HG-induced mesangial cells and accumulation of extracellular matrix via GAS5 SV40 MES-13 cells were transfected with si-control or si-GAS5. Then, SV40 MES-13 cells were divided into NG group, HG group, HG+DGTXT group, HG+DGTXT+si-control group, HG+DGTXT+si-GAS5 group. (A) The proliferation of SV40 MES-13 cells was detected in NG group, HG group, HG+DGTXT group, HG+DGTXT+si-control group, HG+DGTXT+si-GAS5 group. (B) The levels of LN, FN and Col IV were detected in NG group, HG group, HG+DGTXT group, HG+DGTXT+si-control group, HG+DGTXT+si-GAS5 group. ** P<0.01, compared with NG. ##P<0.01, compared with HG. &P<0.01, compared with HG+DGTXT+si-control.

• 

  Figure 6. GAS5 regulated the proliferation of HG-induced mesangial cells and accumulation of extracellular matrix through NF-κB pathway SV40 MES-13 cells were transfected with pcDNA, or pcDNA-GAS5, or pcDNA-GAS5+si-control, or pcDNA-GAS5+si-IKK. Then, SV40 MES-13 cells were divided into NG group, HG group, HG+pcDNA group, HG+pcDNA-GAS5 group, HG+pcDNA-GAS5+si-control group, HG+pcDNA-GAS5+si-IKK group. (A) The proliferation of SV40 MES-13 cells was detected in NG group, HG group, HG+pcDNA group, HG+pcDNA-GAS5 group, HG+pcDNA-GAS5+si-control group, HG+pcDNA-GAS5+si-IKK group. (B) The levels of LN, FN and Col IV were detected in NG group, HG group, HG+pcDNA group, HG+pcDNA-GAS5 group, HG+pcDNA-GAS5+si-control group, HG+pcDNA-GAS5+si-IKK group.** P<0.01, compared with NG. ##P<0.01, compared with HG+pcDNA. &P<0.01, compared with HG+ pcDNA-GAS5+si-control.