The mitochondrial SIRT3 is a major deacetylase protein that regulates important mitochondrial biology, including metabolic pathways, reactive oxygen species detoxification, mitochondrial dynamics, and the mitochondrial unfolded protein response. We found that astragalus aqueous extract increased the activity of SIRT3 in vitro. We further explore the potential antioxidative effects of Astragaloside IV (AST‐IV) and Formononetin (FMR) on induced oxidative stressed AML12 (alpha mouse liver 12) cells. The AML12 cell line was established from healthy hepatocytes from a mouse (CD1 Strain, Line MT42) transgenic for human TFG alpha. We first tested whether AST‐IV or formononetin increases SIRT3 expression in cell model. We cultured AML12 and treated with various concentrations of AST‐IV or formononetin in parallel with untreated control, negative control, and positive control. We found that AST‐IV and formononetin induced SIRT3 expression through Western Blotting (WB) analysis. In addition, we assessed rescue effects of AST‐IV and formononetin on t‐BHP‐induced oxidative injury in AML12 hepatocytes under three separate conditions: pre‐treatment with AST‐IV or formononetin, post‐treatment with AST‐IV or formononetin, and both pre‐treatment and post‐treatment with AST‐IV and formononetin. Through CCK‐8 analysis, we identified that both pre‐treatment and post‐treatment significantly increased cell proliferation on induced‐oxidative stress AML12 cells compared to no treatment and control. Pre‐treatment of AML12 cells before induction of oxidative stress with AST‐IV and formononetin also showed cell proliferation but was less significant compared to AML12 cells that received both pre‐treatment and post‐treatment with AST‐IV or formononetin. Post‐treatment AST‐IV or formononetin after induction of oxidative stress on AML12 cells did not significantly show any difference in cell proliferation compared to non‐treatment and control. Significantly AST‐IV and formononetin showed induction of the SIRT3 protein on tert‐butyl hydroperoxide (t‐BHP) induced oxidative stressed AML12 hepatocytes by WB. Taken together, the increase in cell proliferation is indicative of the antioxidative effects of AST‐IV and formononetin through induction of SIRT3 protein. Further investigation is currently conducted to provide evidence of AST‐IV and formononetin's mechanistic effect on inducing the expression of the SIRT3 protein‐linked downstream pathways for their antioxidative benefit to hepatocytes. The ultimate goal is to utilize these SIRT3 modulators as pharmacologic agents in treating oxidative injury hepatocytes.

Fibroblast plays an important role in keeping heart shape and elasticity as well as maintaining normal cardiac function. The differentiation of fibroblast to myofibroblast is generally considered as an irreversible conversion which represents a critical step in pathogenesis of fibrosis. Transforming growth factor (TGF)‐β1 is a major cytokine that mediates the conversion of fibroblast to myofibroblast, defined by the overly expression of α‐smooth muscle actin (α‐SMA)‐ a biomarker of myofibroblast with little to no appearance of fibroblast specific protein 1 (FSP‐1) – a biomarker of fibroblast. The cellular and molecular mechanisms of myofibroblast differentiation from fibroblasts have been studied extensively. Reversal of myofibroblast differentiation to fibroblasts remains unclear and incompletely understood. Phorbol 12‐myristate 13 acetate (PMA) is involved in multiple cellular functions such as cell growth, differentiation, programmed cell death via protein kinase C (PKC) signaling pathways. To investigate whether PMA dedifferentiates the formed myofibroblasts, NIH 3T3 fibroblasts and human cardiac fibroblast (HCF), cultured in DMEM and fibroblast medium (FM)‐2 respectively, were induced to convert into myofibroblasts in the presence of 2 ng/ml of TGF‐β1 for 24‐ or 48‐hour incubations. Expression of α‐SMA and FSP‐1 in both cell lines was detected by using western blotting and immunofluorescence. Collagen gel contraction induced by cardiac fibroblasts was determined as well. After incubation with TGF‐β1, morphology changes in the shape and the size of NIH 3T3 and HCF cells were observed by the presence of large nuclei and cytoplasm. The levels of expression of α‐SMA were significantly increased whereas expression of FSP‐1 was reduced after 48‐hour incubation with TGF‐β1. NIH 3T3 and HCF cells were then treated with 50 ng/ml of DMSO which used as control groups and with various PMA concentrations (10 ng/ml, 50 ng/ml and 100 ng/ml) for additional 24‐ and 48‐ hour incubations. Chemiluminescence detection of Western blot confirmed the reduction in expression of α‐SMA. The shape and the size of the both cell lines were recovered under the presence of PMA. The results indicated that the reduction in expression of α‐SMA was directly proportional to the concentration of PMA. As PMA concentration increased, the expression of α‐SMA remarkably decreased. PMA also reduced TGF‐β1‐induced collagen gel contraction. These data unambiguously elucidate the reversal of myofibroblast differentiation induced by PMA. Under basic condition, TGF‐β1 induces myofibroblast conversion from fibroblast, and myofibroblast de‐differentiates back into fibroblast in the presence of PMA. Although the mechanism remains to be identified, the novel findings of this study shed light on future development of novel agents to treat fibrotic diseases. Support or Funding Information This project was supported by the Seed Grant from California Northstate University, College of Pharmacy.

Cardiac fibrosis plays an essential role in cardiac pathogenic processes that occur as a result of myocardial infarction or hypertrophic cardiomyopathy. The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in the activation and progression of cardiac fibrosis. TGFβ is one of the essential molecules that promote transition of fibroblasts to myofibroblasts. Reversal of formed myofibroblasts to fibroblasts remains incompletely understood. Phorbol 12-Myristate 13-Acetate (PMA) regulates metabolism and functions of multiple cells via PKC activation mostly. To study effects of PMA on differentiation of de novo formed cardiac myofibroblasts, human cardiac fibroblasts were utilized. Human cardiac fibroblasts (HCF) cultured in fibroblast medium (FM)-2 were converted into myofibroblasts in the presence of 2 ng/mL of TGF-β1 for 48 hours. Expression of α-SMA, the biomarker of myofibroblasts, and FSP1, the biomarker of fibroblasts, was detected using Western blot and immunofluorescence. Collagen gel contraction induced by fibroblasts was determined as well. TGF-β1 increased the expression of α-SMA and reduced the expression of FSP1. Distinct cellular morphology changes in the shape and size of HCF were observed after incubation with TGF-β1 for 48 hours. To investigate effect of PMA on dedifferentiation of formed myofibroblasts, these TGF-β1-pretreated cells were divided into four groups for additional 48 hours incubation: PMA groups (10, 50, and 100 ng/mL) or DMSO (vehicle control). Both 50 and 100 ng/mL of PMA reduced the expression of α-SMA but only 100 ng/mL of PMA increased the expression of FSP1. The shape and size of cells changed after treatment with PMA. PMA also reduced TGF-β1-induced collagen gel contraction (P<0.05, compared to DMSO group). These data indicated that PMA can reverse the differentiation of de novo formed human cardiac myofibroblasts induced by TGF-β1 to fibroblasts and other unidentified type of cells. Although the mechanism of dedifferentiation remains to be identified, the novel finding of this study shed light on future development of agents to treat fibrotic diseases.

The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in the activation and progression of cardiac fibrosis. TGF‐β1 is one of the essential molecules that promotes transition of fibroblasts to myofibroblasts. Reversal of formed myofibroblasts to fibroblasts remains incompletely understood. In our previous studies, Phorbol 12‐Myristate 13‐Acetate (PMA) induces reversal of myofibroblast differentiation via protein kinase C (PKC)‐independent mechanism. Prostaglandin E2 (PGE2) has been shown to reserve differentiation of myofibroblasts of fetal and adult lung fibroblasts. The role of PGE2 in cardiac myofibroblast dedifferentiation remains unknown. Human cardiac fibroblasts were cultured in fibroblast medium (FM)‐2. TGF‐β1 (2ng/mL) was added to FM‐2 for 48 hours to convert fibroblasts into myofibroblasts. PMA (50 ng/mL) or PGE2 (500 nM) was added into cultured cells for 48 hours, respectively. Expression of α‐smooth muscle action (SMA), a biomarker of myofibroblasts, and fibroblast specific protein 1 (FSP‐1), the biomarker of fibroblasts, were detected by using western blotting and immunofluorescence. To explore the involvement of cyclooxygenase 2 (COX‐2)/PGE2 pathway in PMA‐induced reversal of cardiac myofibroblast differentiation, NS‐398, the selective COX‐2 inhibitor, and PF‐04418948, a selective PGE2 receptor antagonist, were applied. Endogenous levels of PGE2 in cardiac myofibroblasts were detected by using Elisa assay kit. TGF‐β1 promoted conversion of cardiac fibroblasts to myofibroblasts as evidenced by increased expression of α‐SMA and reduced expression of FSP‐1. Treatment with PMA dose‐dependently attenuated expression of de novo myofibroblasts. Both NS‐398 and PF‐04418948 exerted no effects on PMA‐induced reversal of cardiac myofibroblasts. Addition of PGE2 into culture medium had no effect on expression of α‐SMA from myofibroblasts. PMA dose‐dependently enhanced formation of PGE2 levels in cardiac myofibroblasts. In conclusion, PMA‐induced reversal of cardiac myofibroblast is independent of activation of COX‐2 and PGE2 pathway. The mechanism in PMA‐induced reversal of cardiac myofibroblasts remains to be further explored.

The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in activation and progression of cardiac fibrosis in heart disease. TGF-β is one of the key cytokines that promotes transition of fibroblasts to myofibroblasts. Dedifferentiation of formed myofibroblasts or reversal of formed myofibroblasts to fibroblasts remains incompletely understood. Prostaglandin E2 (PGE2) has been shown to dedifferentiate human lung myofibroblasts. The role of activation of the COX-2/PGE2 pathway in dedifferentiation of cardiac myofibroblasts remains unknown. Here, we show that phorbol 12-myristate 13-acetate (PMA) but not PGE2 induces dedifferentiation of de novo adult human cardiac myofibroblasts stimulated by TGF-β1 from human cardiac fibroblasts as evidenced by reduced expression of α-smooth muscle actin (α-SMA). PMA remarkably increased endogenous levels of PGE2 in human cardiac myofibroblasts. Pretreatment of myofibroblasts with NS-398, a selective COX-2 inhibitor, and PF-04418948, a selective PGE2 receptor type 2 (EP2) antagonist, had no effect on expression of α-SMA nor abolished the dedifferentiation induced by PMA. Our results indicated that endogenous and exogenous PGE2 has no effects on dedifferentiation of cardiac myofibroblasts. PMA-induced dedifferentiation of cardiac myofibroblast is independent of activation of COX-2 and PGE2 pathway. The mechanism in PMA-induced reversal of cardiac myofibroblasts needs to be explored further.