Background
Hepatic diseases are a aserious problem worldwide, and cause more than 2 million deaths yearly [
1]. Etiologies include viruses, alcohol and non-alcoholic steatohepatitis, among others [
2]. Chronic liver diseases often stimulate hepatic fibrogenic response [
3], which is characterized by excessive synthesis of extracellular matrix (ECM) compounds, loss of parenchymal architecture, inflammation and deposition of scar fibers [
4,
5]. No antifibrotic drugs have been approved for liver diseases. In the liver, hepatic stellate cells (HSCs) play a key role in the development of fibrosis [
6,
7]. In healthy tissues, those cells maintain a quiescent-like phenotype, containing lipid droplets (LDs) that are rich in triglycerides, retinyl esters and cholesterol esters [
8‐
10]. In an injured liver, cells are activated, lose their lipid droplets, increase the production of the ECM elements and transdifferentiate to a more proliferative and fibrogenic state [
11]. The transdifferentiation also induces cellular cytoskeleton changes through the development of prominent cytoplasmic fibers and increased cell size [
12]. This cellular reorganization may facilitate the adhesion, migration and surface remodeling of the cells [
13], as well as changes in several cellular and molecular signaling pathways. However, the mechanistic details that direct cellular behavior are still elusive. Thus, the clarification of molecular routes that support the mechanisms of cellular transdifferentiation could contribute to innovative strategies to treat hepatic fibrosis.
Medicinal plants have been used worldwide to treat hepatic diseases [
14,
15].
Baccharis dracunculifolia D.C. (
Asteraceae family) and
Plectranthus barbatus Andrews (
Lamiaceae family) are two well-known species reported in the literature for the treatment of liver pathologies and widely used in Brazil as medicinal herbal teas [
16,
17]. The plant leaves are rich in several diterpenoids, phenolic compounds and essential oils [
18,
19], which supports their anti-inflammatory, antioxidant, and hepatoprotective activities [
20‐
23]. In addition, forskolin, the most studied constituent of the
P. barbatus, was demonstrated to have anti-fibrotic properties by regulating cyclic AMP (cAMP) activator and controlling triglyceride metabolism [
23‐
25]. Likewise, the medicinal plant
B. dracunculifolia has a protective effect in the liver [
26], as its byproducts, including Brazilian green propolis has been described as a modulator of inflammation and fibrogenesis [
27,
28]. Therefore, based on their properties, it is relevant to understand the molecular and cellular routes by which these plants modulate liver diseases.
Because the HSCs are extremely responsive to liver injury, the present study investigated cellular and molecular effects of
Baccharis dracunculifolia and
Plectranthus barbatus leaf extracts in LX-2 cells cultivated under activated conditions. LX-2 is a human immortalized hepatic stellate cell line [
29] that has been widely used to study the processes of stellate cell transdifferentiation and fibrogenesis. The effects of the plant extracts in LX-2 were analyzed by measuring their cytotoxicity and genotoxicity, as well as by assessing their effects on cellular morphology, lipid droplet distribution and metabolism and gene expression for fibrotic markers that are relevant to cellular transdifferentiation [
18].
Under the investigated concentrations of the plant extracts no deleterious effects on LX-2 metabolism, such as toxicity, genotoxicity, or apoptosis were observed. Moreover, the plant extracts induced changes in actin filament distribution of activated LX-2, despite no relevant effect on cellular markers of transdifferentiation. On the other hand, consistent effects in cellular lipid metabolism were observed in plant extract-treated cells, supporting the presumed activity of plant extracts in hepatic metabolism. In addition, the combined results suggested an innovative function of the plants in controlling LX-2 retinoid metabolism, which may exert protective effects upon liver injuries and collaborates as a co-factor element in the controlling cellular transdifferentiation and fibrosis processes [
30].
Discussion
The search for alternative methods to control hepatic diseases has intensified, and the pharmacologic effects of medicinal plants have become the main focus of several investigations. In the present study, different cytotoxicity levels were observed for
P. barbatus and
B. dracunculifolia extracts on the activated LX-2 cells (Fig.
1). In addition, the comet assays demonstrated that the extracts had little genotoxic effects on LX-2 cells, as no significant damage to the DNA molecules was observed. Comet assays found relevant information on DNA integrity [
37] and its correlation with the possible activation of cell death mechanisms [
38]. The cells treated with
B. dracunculifolia demonstrated decreased DNA damage compared to the non-treated activated cultures. These results corroborate Munari et al. (2009) [
39], who studied the antigenotoxic effects of
B. dracunculifolia ethanol extract on lung fibroblasts of Chinese hamsters (V 79 cells) exposed to 200 μM of methyl methanesulfonate (MMS) which confirmed the genoprotective capability of the extract. Moreover, our results are in accordance with Roberto et al. (2016) [
21], who observed the hepatoprotective capability of
B. dracunculifolia metabolic products against genotoxic agents [
40]. According to Tapia et al. (2004) and Resende et al. (2007), the protective effects of some
Baccharis species can be attributed to the antioxidant activities of their active phenolic components [
41,
42].
For proapoptotic signaling (Fig.
1c), the
B. dracunculifolia extract reduced the transcriptional levels of the analyzed genes. Some authors have reported that the extract can modulate the expression levels of proapoptotic genes, such as
caspase 3, as observed in this study [
43,
44], confirming the beneficial and hepatoprotective effects of the extract, which presents antioxidant and anti-inflammatory molecules [
26,
27,
45]. In our study, high expression levels of the antiapoptotic gene
bcl-2 were observed in cells treated with
P. barbatus, which can facilitate the progression of hepatic fibrosis [
46]. According to the literature, high bcl-2 signaling can increase HSC resistance to other proapoptotic stimuli, worsening the fibrotic condition [
47].
The transdifferentiation effects on LX-2 cells alter the morphology of the cytoskeleton, which cause the appearance of more polymerized actin filaments in the cytoplasm [
12]. In the present study, the treatment with both extracts increased the number of polymerized filaments in the cytoskeleton in the cells (Fig.
2). In addition,
P. barbatus increased the expression of
α-SMA, a classic HSC transdifferentiation marker [
48,
49]. Considering the hepatic fibrosis environment, excessive number of thicker and more diffuse F-actin filaments can lead to an increase in HSC profibrotic and proinflammatory signaling [
13,
50‐
53], which alters the homeostatic cellular environment.
During the HSC activation, the lipid metabolism is altered, and an expressive loss of intracellular droplets was observed (Fig.
3).
B. dracunculifolia is not capable of modulating lipid droplet assembly. On the other hand,
P. barbatus caused the droplet packing closer to the pattern found in quiescent-like cellular phenotype. Considering these data, we emphasized the relevance of the gene
crabp-2 (Fig.
3), which plays a role in the transport and nuclear signaling of retinoic acid, a type of lipid present in the droplets [
10,
54]. In our experiment, this gene has minimum expression levels in LX-2 cells treated with
B. dracunculifolia, while in those treated with
P. barbatus, expression levels were significantly increased, which favors the LD formation. In addition, the transcriptional levels of relevant genes correlated with the lipid droplets assembling were affected by the plant extracts. The gene
bscl-2, which drives the synthesis of the enzyme BSCL2, presented increased expression in the cells treated with
P. barbatus extract. This enzyme is found in the endoplasmic reticulum, functioning in association with the DGAT enzymes for assembling and maturation of the lipid droplets [
55,
56]. The increased expression of
bscl-2 suggests the presence of a higher concentration of active lipids available for lipid droplet assembling and a participative activity of the enzyme in this cellular process activated by the
P. barbatus extract.
To corroborated the effect of the plant extracts in general lipid metabolism, FT-IR analyses were performed (Fig.
4a) and demonstrated increased lipid levels in the
P. barbatus-treated cells compared to the other treatments. Furthermore, the decreased transcriptional levels of the gene
atgl suggests reduced activity of the enzyme ATGL (Fig.
4b), which has the capability of hydrolyzing triacylglycerols and retinol esters in the HSCs [
57], facilitating the accumulation of lipids in these cells. The transcriptional levels of DGAT-1 are higher in the cultures treated with
B. dracunculifolia than the other treatments. The DGAT-1 is directly associated with the synthesis of triacylglycerols from diacylglycerols [
58]. In the liver, this enzyme acts on the esterification of exogenous fatty acids to glycerol, which can be used as an energy source, avoiding its accumulation [
59,
60]. This molecular mechanism justifies the lower concentration of lipids observed in the FT-IR assays in the cells incubated with
B. dracunculifolia, compared to the results found in
P. barbatus-treated
cells (Fig.
4a). In addition, the increased transcriptional levels of ACSL4, CS and ATGL in the cells treated with
B. dracunculifolia extract (Fig.
4b), indicated the utilization of metabolites as energy source in mitochondrial oxidation processes [
61,
62]. Combined, these results demonstrated that the extracts activated differential biochemical processes in the LX-2 cells; however, such processes were independent of the transcription factors classically pointed to as lipid metabolism modulators [
63,
64].
Generally, lipid metabolism helps to support the cellular effects of medicinal plant extracts. In a cell, it is assumed that the increase in total lipids contributes to the synthesis of ECM compounds and to the cell transdifferentiation process, enabling the rearrangement of the organelles and the synthesis of membranes when the cells have their phenotype activated [
8]. However, our results suggested that the treatment with
B. dracunculifolia were able to maintain lipid/retinoid homeostasis in the activated cultures, in agreement with a previous study [
21]. In comparison with the results of the activated cultures, the addition of
B. dracunculifolia extract reduced the expression of genes related to apoptosis and maintained the lipid metabolism similar to that found in the activated LX-2 cultures, suggesting a direct action of the plant on the protective mechanisms, without controlling/reverting the pro-fibrotic condition of the HSCs cultures. In parallel, the
P. barbatus extracts did not extremely alter the expression of genes correlated with the proapoptotic process (Fig.
1). However, a reorganization of the actin filaments and increased transcriptional level of relevant markers of cellular activation occured (Fig.
2), suggesting profibrotic signaling activation, which corroborates studies that point to
P. barbatus toxicity when used in high concentrations [
65,
66]. Moreover, the extract also acted directly on cellular lipid metabolism, reinforcing popular culture, which uses the extracts as a liver fat metabolizer [
16,
67,
68]. The reemergence of lipid droplets in the cells treated with
P. barbatus may be a response to the toxicity of free fatty acids, but this requires further investigation [
69].
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