Shenweifang-containing serum inhibits transforming growth factor-β1 induced myofibroblast differentiation in normal rat kidney interstitial fibroblast cell

Abstract

Objective: To investigate the efficacy of Shenweifang (SWF)-containing serum on transforming growth factor (TGF)-β1-induced fibroblast-myofibroblast transition in normal rat kidney interstitial fibroblast cells (NRK-49F).

Methods: Sprague-Dawley rats were gavaged with one of five solutions: (a) saline; (b) saline plus low-dose SWF; (c) saline plus medium-dose SWF; (d) saline plus highdose SWF; and (e) saline plus valsartan. NRK-49F cells were treated with TGF-β1 and cultured using serum from the gavaged rats.

Results: TGF-β1 treatment increased the expression of α-smooth muscle actin, proliferating cell nuclear antigen, collagen I, Smad3, mitogen-activated protein kinase (MAPK) 10, and c-Jun N-terminal kinase (JNK) 3 and induced abnormalities in cell morphology, cell cycle progression, and cell proliferation.

Conclusions: SWF- or valsartan-containing serum corrected (or partially corrected) TGF-β1-induced abnormal changes in this in vitro system. SWF-containing serum reversed abnormalities in morphology, cell cycle progression, and proliferation in TGF-β1-treated NRK49F cells, probably by blocking the TGF-β1/Smads and TGF-β1/MAPK/JNK pathways.

Keywords: Fibrosi; JNK mitogenactivated protein; Kinases drug-containing serum; Shenweifang; Smad proteins; mitogen-activated protein kinase; Transforming growth factor beta1.

Figure 1. Effect of Shenweifang (SWF)-containing serum on the cell cycle distribution of transforming growth factor (TGF)-β1-treated normal rat kidney interstitial fibroblast cells (NRK-49F)

As A and B shows control group, untreated cells (cultured in 10% fetal bovine serum). TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% normal rat serum (rats were gavaged saline). Low-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% low-dose SWF rat serum (rats were gavaged low-dose SWF). Medium-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% medium-dose SWF rat serum (rats were gavaged medium-dose SWF). High-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% high-dose SWF rat serum (rats were gavaged high-dose SWF). Valsartan serum+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% valsartan-containing rat serum (rats were gavaged valsartan). n = 3. ^aP ˂ 0.01 compared with control cells; ^bP < 0.01 compared with TGF-β1-treated cells.

Figure 2. Effect of Shenweifang (SWF)-containing serum on the protein expression of α-smooth muscle actin (α-SMA), fibronectin, and proliferating cell nuclear antigen (PCNA) in transforming growth factor (TGF)-β1-treated normal rat kidney interstitial fibroblast cells (NRK-49F)

Control group, untreated cells (cultured in 10% fetal bovine serum). TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% normal rat serum (rats were gavaged saline). Low-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% low-dose-SWF rat serum (rats were gavaged low-dose SWF). Medium-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% medium-dose-SWF rat serum (rats were gavaged medium-dose SWF). High-dose SWF + TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% high-dose-SWF rat serum (rats were gavaged high-dose SWF). Valsartan serum + TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% valsartan-containing rat serum (rats were gavaged valsartan).

Figure 3. Effect of Shenweifang (SWF)-containing serum on the mRNA expression of α-smooth muscle actin (α-SMA), fibronectin, proliferating cell nuclear antigen (PCNA), and collagen Ⅰ in transforming growth factor (TGF)-β1-treated NRK-49F cells

A: α-SMA; B: fibronectin; C: PCNA; D: collagen Ⅰ. Control group, untreated cells (cultured in 10% fetal bovine serum). TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% normal rat serum (rats were gavaged saline). Low-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% low-dose SWF rat serum (rats were gavaged low-dose SWF). Medium-dose SWF + TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% medium-dose SWF rat serum (rats were gavaged medium-dose SWF). High-dose SWF + TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% high-dose SWF rat serum (rats were gavaged high-dose SWF). Valsartan serum + TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% valsartan-containing rat serum (rats were gavaged valsartan). n = 3. ^aP ˂ 0.01, compared with control cells; ^bP < 0.01, compared with TGF-β1-treated cells. SWF: Shenweifang; α-SMA: α-smooth muscle actin; PCNA: proliferating cell nuclear antigen; TGF-β1: transforming growth factor-β1.

Figure 4. Effect of Shenweifang (SWF)-containing serum on the mRNA expression of Smad3 and MAPK-10 in transforming growth factor (TGF)-β1-treated NRK-49F cells

Control group, untreated cells (cultured in 10% fetal bovine serum). TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% normal rat serum (rats were gavaged saline). Low-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% low-dose SWF rat serum (rats were gavaged low-dose SWF). Medium-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% medium-dose SWF rat serum (rats were gavaged medium-dose SWF). High-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% high-dose-SWF rat serum (rats were gavaged high-dose SWF). Valsartan serum+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% valsartan-containing rat serum (rats were gavaged valsartan). n = 3. ^aP ˂ 0.01, compared with control cells; ^bP < 0.01, ^cP < 0.05, compared with TGF-β1-treated cells.

Figure 5. Effect of Shenweifang (SWF)-containing serum on the protein expression of α-smooth muscle actin (α-SMA), fibronectin, proliferating cell nuclear antigen (PCNA), and collagen Ⅰ in transforming growth factor (TGF)-β1-treated NRK-49F cells

Control group, untreated cells (cultured in 10% fetal bovine serum). TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% normal rat serum (rats were gavaged saline). Low-dose SWF+TGF-β1 group (low-dose group), cells treated with TGF-β1 (10 ng/mL) and 10% low-dose SWF rat serum (rats were gavaged low-dose SWF). Medium-dose SWF+TGF-β1 group (medium-dose group), cells treated with TGF-β1 (10 ng/mL) and 10% medium-dose SWF rat serum (rats were gavaged medium-dose SWF). High-dose SWF+TGF-β1 group (high-dose group), cells treated with TGF-β1 (10 ng/mL) and 10% high-dose SWF rat serum (rats were gavaged high-dose SWF). Valsartan serum+TGF-β1 group (Val group), cells treated with TGF-β1 (10 ng/mL) and 10% valsartan-containing rat serum (rats were gavaged valsartan). n = 3. ^aP ˂ 0.01, compared with control cells; ^bP < 0.01, ^cP < 0.05, compared with TGF-β1-treated cells.

Figure 6. Effect of Shenweifang (SWF)-containing serum on the protein expression of Smad2/3, phosphorylated Smad3 (p-Smad3), c-Jun N-terminal kinase (JNK), and JNK-3 in transforming growth factor (TGF)-β1-treated NRK-49F cells

Control group, untreated cells (cultured in 10% fetal bovine serum). TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% normal rat serum (rats were gavaged saline). Low-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% low-dose SWF rat serum (rats were gavaged low-dose SWF). Medium-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% medium-dose SWF rat serum (rats were gavaged medium-dose SWF). High-dose SWF+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% high-dose SWF rat serum (rats were gavaged high-dose SWF). Valsartan serum+TGF-β1 group, cells treated with TGF-β1 (10 ng/mL) and 10% valsartan-containing rat serum (rats were gavaged valsartan). n = 3. ^aP ˂ 0.01, compared with control cells; ^bP < 0.05, ^cP < 0.01, compared with TGF-β1-treated cells.