J Mol Med DOI 10.1007/s00109-015-1283-1
ORIGINAL ARTICLE
Berberine inhibits Smad and non-Smad signaling cascades and enhances autophagy against pulmonary fibrosis Palanivel Chitra 1 & Gowrikumar Saiprasad 1 & Ramar Manikandan 2 & Ganapasam Sudhandiran 1
Received: 12 May 2014 / Revised: 13 February 2015 / Accepted: 31 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative lung disorder of unknown aetiology. Transforming growth factor-β1 (TGF-β1)-mediated Smad and non-Smad signaling cascades are considered as central players in accelerating pulmonary fibrosis. We earlier reported berberine’s amelioration against TGF-β1-mediated pro-fibrotic effects in bleomycininduced pulmonary fibrosis. The present study aimed to determine the regulatory role of berberine on abrogated Smad 2/ 3 and FAK-dependent PI3K/Akt-mTOR signaling cascades in bleomycin-induced pulmonary fibrosis. Male Wistar rats were subjected to single intratracheal instillation of bleomycin (2.5 U/kg) on day 0, and berberine treatments were provided in either preventive or therapeutic modes, respectively. Berberine mitigated the elevated expression of fibrotic markers, α-smooth muscle actin (α-SMA), fibronectin, collagens I and III and reversed bleomycin-induced ultrastructural alterations in the lungs. Berberine inhibited the bleomycininduced raise in p-Smad 2/3 and enhanced Smad 7 expression. Berberine blocked the activation of FAK and PI3K/Akt against bleomycin-induced dysregulation, with subsequent raise in PTEN expression. In addition, by inhibiting pmTOR, berberine stimulated autophagy as evidenced by Electronic supplementary material The online version of this article (doi:10.1007/s00109-015-1283-1) contains supplementary material, which is available to authorized users. * Ganapasam Sudhandiran [email protected]; [email protected] 1
Department of Biochemistry, Cell Biology Laboratory, University of Madras, Guindy Campus, Chennai, Tamil Nadu 600025, India
2
Department of Zoology, University of Madras, Guindy Campus, Chennai, Tamil Nadu 600025, India
increase in Beclin-1, LC3-II levels with enhanced autophagosome formation. Cumulatively, through targeted inhibition of dysregulated Smad and FAK-dependent PI3K/AktmTOR signaling axis, berberine attenuated the fibrotic insults of bleomycin. Key message & Berberine inhibits Smad 2/3 activation and enhances Smad 7 in bleomycin-induced rat lungs. & Bleomycin-induced activation of FAK is inhibited by berberine. & Berberine inhibits bleomycin-induced activation of PI3K/ Akt cascade. & Berberine inhibits mTOR activation to enhance autophagy and suppresses fibrotic markers. Keywords Pulmonary fibrosis . Smad . FAK . PI3K . mTOR . Autophagy
Introduction Pulmonary fibrosis is a heterogeneous disorder of lung interstitium which involves continuous epithelial injury with abnormal wound healing and excessive extracellular matrix depositions. The incidence and annual mortality rate of pulmonary fibrosis seems to be increasing over the past two decades. Despite improvements in diagnostic approaches, no convincing or effective therapies exist for pulmonary fibrosis, which makes the median survival from diagnosis to be 2–3 years [1]. Though, critical event’s underlying the pathogenesis of pulmonary fibrosis has not been completely explored, it seems to be accompanied with transitions of alveolar epithelial cells to proliferative mesenchymal cell populations (fibroblasts/ myofibroblasts), together with extracellular matrix (ECM)
J Mol Med
accumulations. The molecular pathways engaged in regulating these pathogenic proceedings are complex and likely multi-factorial. The present treatment options targeting a specific molecule/event and its associated side effects have been typically failed in fibrosis prevention or therapy. Therefore, it is important to design a strategy that could simultaneously target multiple cellular signaling events which will be effective in resolving pulmonary fibrosis [2]. Perhaps, the potent action of naturally derived phytochemicals and their targeted attenuation of exaggerated signaling pathways against fibrosis have been proved [3]. Recently, we demonstrated the antifibrotic potential of one such multi-targeted alkaloid berberine against bleomycin-induced oxidative stress and inflammatory responses in a biphasic manner [4]. However, its efficacy over the critically activated signaling events in bleomycin-induced pulmonary fibrosis remains unstudied. This study is focused to determine the regulatory mechanism of berberine over the dysregulated signaling cascades involved in bleomycininduced pulmonary fibrosis. Several growth factors and cytokines regulate epithelial to mesenchymal transition (EMT), fibroblast proliferation and ECM deposition in the lung which leads to varying degree of pulmonary fibrosis. The activation of transforming growth factor beta (TGF-β), a pleiotropic cytokine, remains a prime focus in fibrotic lung disease [5]. Smad proteins particularly Smad 2/3 are fundamental in executing TGF-β-mediated profibrotic events including secretion of pro-fibrotic cytokines and ECM proteins [6]. In addition to Smad-dependent mechanism, TGF-β1 also signals many Smad-independent pathways, among which the activation of FAK and PI3K/Akt cascade is considered to be vital in mediating pulmonary fibrosis progression [7]. The inhibitors of these kinases have proved to be an effective anti-fibrotic therapy for pulmonary fibrosis [8]. Also, the activity of phosphatase and tensin homolog (PTEN), a dual lipid/protein phosphatase and the major negative regulator of PI3K/Akt, is known to be impaired in pulmonary fibrosis [9]. Impairment of autophagy, a homeostatic mechanism, represents a novel venture for the promotion of pulmonary fibrosis [10]. Moreover, the intracellular pathway PI3K/Akt is known to suppress autophagy via its downstream mTOR [11]. Strategies to improve autophagic response are proven to play a vital role in degrading the abnormally deposited matrix components and recognized as key phenomena to resolve/inhibit fibroblastic disarray and pulmonary fibrosis [12]. Thus, interventions aimed at eliminating Smad aberrations and interrupting the activation of FAK and PI3K/AktmTOR signaling cascade represents an attractive and promising approach for a potent anti-fibrotic agent against pulmonary fibrosis. The bleomycin-induced rodent model of pulmonary fibrosis is an excellent tool to investigate novel treatments for pulmonary fibrosis which can reiterate the complex
Fig. 1 a mRNA profiles of α-SMA, fibronectin and collagens I and III using RT-PCR from the lung tissues of control and experimental groups of animals. b Gene expressions of α-SMA, fibronectin and collagens I and III normalized to GAPDH are presented as “fold change” as compared with the control group. c Immunoblotting analysis of α-SMA in the lung tissues of control and experimental groups of animals. d Protein expression of α-SMA normalized to β-actin is presented as “fold change” as compared with the control group. e Representative photographs for the immunohistochemical staining of α-SMA. The colour code for positive expression is brown and the counter stain is blue. Control: lung tissue sections of control animals showing nominal expression of α-SMA. BLM: bleomycin-induced animals showing greater expression of α-SMA compared to controls. Lung tissue sections of berberine-treated animals at both preventive [BLM + BBR (1–14 days)] as well as therapeutic mode [BLM + BBR (14–28 days)] showing markedly decreased immunoreactivity of α-SMA. BBR: lung sections of berberine-alone-administered animals showing negligible expressions of α-SMA similar to the control. Black arrows indicate αSMA expression within the fibroblastic foci. Red arrows represent αSMA expression in hyperplasic alveolar epithelial cells overlying areas of fibroblastic foci. The images shown are representative of three separate experiments. The corresponding graph represents the average number of α-SMA positive cells across 20 randomly selected fields. Magnification, 40×; scale bar, 25 μm). Each value is expressed as mean±S.D. of three independently performed experiments (n=3). Hypothesis testing method included one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test. Results are statistically significant at p
ORIGINAL ARTICLE
Berberine inhibits Smad and non-Smad signaling cascades and enhances autophagy against pulmonary fibrosis Palanivel Chitra 1 & Gowrikumar Saiprasad 1 & Ramar Manikandan 2 & Ganapasam Sudhandiran 1
Received: 12 May 2014 / Revised: 13 February 2015 / Accepted: 31 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Idiopathic pulmonary fibrosis (IPF) is a fibroproliferative lung disorder of unknown aetiology. Transforming growth factor-β1 (TGF-β1)-mediated Smad and non-Smad signaling cascades are considered as central players in accelerating pulmonary fibrosis. We earlier reported berberine’s amelioration against TGF-β1-mediated pro-fibrotic effects in bleomycininduced pulmonary fibrosis. The present study aimed to determine the regulatory role of berberine on abrogated Smad 2/ 3 and FAK-dependent PI3K/Akt-mTOR signaling cascades in bleomycin-induced pulmonary fibrosis. Male Wistar rats were subjected to single intratracheal instillation of bleomycin (2.5 U/kg) on day 0, and berberine treatments were provided in either preventive or therapeutic modes, respectively. Berberine mitigated the elevated expression of fibrotic markers, α-smooth muscle actin (α-SMA), fibronectin, collagens I and III and reversed bleomycin-induced ultrastructural alterations in the lungs. Berberine inhibited the bleomycininduced raise in p-Smad 2/3 and enhanced Smad 7 expression. Berberine blocked the activation of FAK and PI3K/Akt against bleomycin-induced dysregulation, with subsequent raise in PTEN expression. In addition, by inhibiting pmTOR, berberine stimulated autophagy as evidenced by Electronic supplementary material The online version of this article (doi:10.1007/s00109-015-1283-1) contains supplementary material, which is available to authorized users. * Ganapasam Sudhandiran [email protected]; [email protected] 1
Department of Biochemistry, Cell Biology Laboratory, University of Madras, Guindy Campus, Chennai, Tamil Nadu 600025, India
2
Department of Zoology, University of Madras, Guindy Campus, Chennai, Tamil Nadu 600025, India
increase in Beclin-1, LC3-II levels with enhanced autophagosome formation. Cumulatively, through targeted inhibition of dysregulated Smad and FAK-dependent PI3K/AktmTOR signaling axis, berberine attenuated the fibrotic insults of bleomycin. Key message & Berberine inhibits Smad 2/3 activation and enhances Smad 7 in bleomycin-induced rat lungs. & Bleomycin-induced activation of FAK is inhibited by berberine. & Berberine inhibits bleomycin-induced activation of PI3K/ Akt cascade. & Berberine inhibits mTOR activation to enhance autophagy and suppresses fibrotic markers. Keywords Pulmonary fibrosis . Smad . FAK . PI3K . mTOR . Autophagy
Introduction Pulmonary fibrosis is a heterogeneous disorder of lung interstitium which involves continuous epithelial injury with abnormal wound healing and excessive extracellular matrix depositions. The incidence and annual mortality rate of pulmonary fibrosis seems to be increasing over the past two decades. Despite improvements in diagnostic approaches, no convincing or effective therapies exist for pulmonary fibrosis, which makes the median survival from diagnosis to be 2–3 years [1]. Though, critical event’s underlying the pathogenesis of pulmonary fibrosis has not been completely explored, it seems to be accompanied with transitions of alveolar epithelial cells to proliferative mesenchymal cell populations (fibroblasts/ myofibroblasts), together with extracellular matrix (ECM)
J Mol Med
accumulations. The molecular pathways engaged in regulating these pathogenic proceedings are complex and likely multi-factorial. The present treatment options targeting a specific molecule/event and its associated side effects have been typically failed in fibrosis prevention or therapy. Therefore, it is important to design a strategy that could simultaneously target multiple cellular signaling events which will be effective in resolving pulmonary fibrosis [2]. Perhaps, the potent action of naturally derived phytochemicals and their targeted attenuation of exaggerated signaling pathways against fibrosis have been proved [3]. Recently, we demonstrated the antifibrotic potential of one such multi-targeted alkaloid berberine against bleomycin-induced oxidative stress and inflammatory responses in a biphasic manner [4]. However, its efficacy over the critically activated signaling events in bleomycin-induced pulmonary fibrosis remains unstudied. This study is focused to determine the regulatory mechanism of berberine over the dysregulated signaling cascades involved in bleomycininduced pulmonary fibrosis. Several growth factors and cytokines regulate epithelial to mesenchymal transition (EMT), fibroblast proliferation and ECM deposition in the lung which leads to varying degree of pulmonary fibrosis. The activation of transforming growth factor beta (TGF-β), a pleiotropic cytokine, remains a prime focus in fibrotic lung disease [5]. Smad proteins particularly Smad 2/3 are fundamental in executing TGF-β-mediated profibrotic events including secretion of pro-fibrotic cytokines and ECM proteins [6]. In addition to Smad-dependent mechanism, TGF-β1 also signals many Smad-independent pathways, among which the activation of FAK and PI3K/Akt cascade is considered to be vital in mediating pulmonary fibrosis progression [7]. The inhibitors of these kinases have proved to be an effective anti-fibrotic therapy for pulmonary fibrosis [8]. Also, the activity of phosphatase and tensin homolog (PTEN), a dual lipid/protein phosphatase and the major negative regulator of PI3K/Akt, is known to be impaired in pulmonary fibrosis [9]. Impairment of autophagy, a homeostatic mechanism, represents a novel venture for the promotion of pulmonary fibrosis [10]. Moreover, the intracellular pathway PI3K/Akt is known to suppress autophagy via its downstream mTOR [11]. Strategies to improve autophagic response are proven to play a vital role in degrading the abnormally deposited matrix components and recognized as key phenomena to resolve/inhibit fibroblastic disarray and pulmonary fibrosis [12]. Thus, interventions aimed at eliminating Smad aberrations and interrupting the activation of FAK and PI3K/AktmTOR signaling cascade represents an attractive and promising approach for a potent anti-fibrotic agent against pulmonary fibrosis. The bleomycin-induced rodent model of pulmonary fibrosis is an excellent tool to investigate novel treatments for pulmonary fibrosis which can reiterate the complex
Fig. 1 a mRNA profiles of α-SMA, fibronectin and collagens I and III using RT-PCR from the lung tissues of control and experimental groups of animals. b Gene expressions of α-SMA, fibronectin and collagens I and III normalized to GAPDH are presented as “fold change” as compared with the control group. c Immunoblotting analysis of α-SMA in the lung tissues of control and experimental groups of animals. d Protein expression of α-SMA normalized to β-actin is presented as “fold change” as compared with the control group. e Representative photographs for the immunohistochemical staining of α-SMA. The colour code for positive expression is brown and the counter stain is blue. Control: lung tissue sections of control animals showing nominal expression of α-SMA. BLM: bleomycin-induced animals showing greater expression of α-SMA compared to controls. Lung tissue sections of berberine-treated animals at both preventive [BLM + BBR (1–14 days)] as well as therapeutic mode [BLM + BBR (14–28 days)] showing markedly decreased immunoreactivity of α-SMA. BBR: lung sections of berberine-alone-administered animals showing negligible expressions of α-SMA similar to the control. Black arrows indicate αSMA expression within the fibroblastic foci. Red arrows represent αSMA expression in hyperplasic alveolar epithelial cells overlying areas of fibroblastic foci. The images shown are representative of three separate experiments. The corresponding graph represents the average number of α-SMA positive cells across 20 randomly selected fields. Magnification, 40×; scale bar, 25 μm). Each value is expressed as mean±S.D. of three independently performed experiments (n=3). Hypothesis testing method included one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test. Results are statistically significant at p
