Wedelolactone from Eclipta alba inhibits lipopolysaccharide-enhanced cell proliferation of human renal mesangial cells via NF-κB signaling pathway

Abstract

Mesangial cells of glomerulus which could produce and degrade several ECMs, take part in the repair and update of mesangial matrix and GBM, regulate glomerular filtration rate, secret cytokines and phagocytose immune complexes contribute a lot to physiological functions and pathological reactions of glomerular. There inflammation response of abnormal proliferation induced by LPS could lead to renal damage. Herein, wedelolactone, an active chemical constituent extracted from leaves of Eclipta alba, was used to explore if it could be an effective inhibitor of the proliferative response of HRMCs. The effects of different concentration wedelolactone on the secretion of cytokines, cell viability and NF-κB pathway were all detected by qPCR, western blotting and ELISA. The results indicated that wedelolactone could inhibit the abnormal proliferation of HRMCs via regulating the activity of several key members of NF-κB signaling pathway.

Keywords: NF-κB signaling pathway; Wedelolactone; cell proliferation; renal mesangial cells.

Figure 1

Effects of LPS on HRMCs viability and the expression of TNF-α and IL-1β. A: The viability of HRMCs incubated with different concentration of LPS for 24 h was determined by CCK-8 assay. B: The supernatant level of TNF-α in HRMCs incubated with different concentration of LPS for 24 h was determined by ELISA assay. C: The supernatant level of IL-1β in HRMCs incubated with different concentration of LPS for 24 h was determined by ELISA assay. Data represent mean ± S.E.M. (n=3).

Figure 2

Effect of wedelolactone on the cell viability of normal HRMCs. The viability of healthy HRMCs treated with different concentrations of wedelolactone was detected via CCK-8 assay. Data represent mean ± S.E.M. (n=3).

Figure 3

Effect of wedelolactone on the enhanced proliferation of LPS-induced HRMCs. Cell viability of LPS-induced HRMCs treated with different concentrations (no more than 20 μmol/l) of wedelolactone for 1 h was determined by CCK-8 assay. Data represent mean ± S.E.M. (n=3).

Figure 4

Effects of low concentration wedelolactone on LPS-mediated production of inflammatory cytokines in HRMCs. A: The supernatant level of TNF-α in LPS-induced HRMCs incubated with low concentrations of wedelolactone for 1 h was determined by ELISA assay. B: The supernatant level of IL-1β in LPS-induced HRMCs treated with low concentrations of wedelolactone for 1 h was determined by ELISA assay. C: The supernatant level of NO in LPS-induced HRMCs treated with low concentrations of wedelolactone for 1 h was determined by Griess assay. Data represent mean ± S.E.M. (n=3).

Figure 5

Effect of wedelolactone on LPS-enhanced DNA binding activity of NF-κB p65. The DNA binding activity of NF-κB p65 in LPS-induced HRMCs treated with different concentrations of wedelolactone was detected using the Trans AMTM NF-κB p65 kit. Data represent mean ± S.E.M. (n=3). ***P<0.001.

Figure 6

Effects of wedelolactone on the mRNA and protein expression levels of IκBα, p-IκBα and IKKβ in LPS-induced HRMCs. A: Relative mRNA expression of IκBα in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by qPCR. B: Relative mRNA expression of IKKβ in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by qPCR. C: Relative protein expression of IκBα in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by western blotting. D: Relative protein expression of IKKβ in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by western blotting. E: Relative protein expression of p-IκBα in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by western blotting. Data represent mean ± S.E.M. (n=3) *P<0.05.

Figure 7

Effects of wedelolactone on the mRNA and protein expression levels of UBE1, UBC5 and E3RS IκB in LPS-induced HRMCs. A: Relative mRNA expression of UBE1 in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by qPCR. B: Relative mRNA expression of UBC5 in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by qPCR. C: Relative mRNA expression of E3RS IκB in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by qPCR. D: Protein expression of UBE1 in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by ELISA assay. E: Protein expression of UBC5 in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by ELISA assay. F: Protein expression of E3RS IκB in LPS-induced HRMCs treated with different concentrations of wedelolactone was measured by ELISA assay. Data represent mean ± S.E.M. (n=3) *P<0.05.