To the best of our knowledge, this project is the first report describing the effect of genistein treatment in the lung of ovariectomized rats with focus on its recovering role in the fibrosis and getting over the inflammation. These findings are novel and have implications in either understanding the pathogenesis of menopause and its treatment. In current study, genistein treatment significantly ameliorated estrogen deficiency induced alteration in the expression of TGFβ1, IL1β, MMP2, ERK, caspase3 and Bcl-2 in the studied groups.
Loss of anti-fibrotic, anti- inflammatory and anti- apoptotic effects of estrogen beginning with menopause is theorized to be responsible for occurrence of pulmonary disease. Thus given that, ovariectomy can cause inflammation and fibrosis [1]. Estrogen deficiency resulted in an increase in TGFβ1 gene transcription in oophorectomized female rabbits [16]. Of note, increase in TGFβ1 mRNA has been shown in fibrotic human lungs [17].
The important role of estrogen in matrix remodeling has been established in the recent years. Previous studies have demonstrated that estrogen replacement can reduce collagen deposition in the lung of OVX rats [1]. Accordingly, estrogen continues to provide a protective effect against cardiac fibrosis [18]. However, there are some contradictory reports indicating that, estrogen can enhance fibrogenesis in a model of fibrotic lung disease [19].
Activation of MAPK/ Erk pathway exerts an important role in fibrosis through regulating the matrix synthesis and/or fibroblast into myofibroblast trans- differentiation [20]. Recent studies have also revealed a new molecular mechanism involved in the MAPK/ Erk pathway by TGFβ1. Thus, activation of ERK1/2 and MAPK pathway by TGFβ1 has been proposed in dermal fibroblast cells [21]. There is increasing evidence that epithelial mesenchymal transition (EMT) has a major role in the progression of pulmonary fibrosis [22]. Also, it has been shown that activation of the Ras/ Erk MAPK signaling pathway might be responsible for EMT induced by TGFβ1 [23].
Several researches suggest that aging associated with a morphological change in the lung alveolar cells [24]. Repetitive alveolar injury resulting in pulmonary fibrosis has been noted to have serious histological abnormality such as denuded basal lamina [25]. In accordance with the last studies, we found significant morphological alterations in alveolar cells characteristic by the alveolar epithelium deformity in the lung of ovariectomized rats.
More studies have highlighted the role of IL1β, as an inflammatory marker, in the progression of lung fibrosis [26]. Evidence will be presented indicating the potential role of IL1β in lung injury and inflammation [27]. Of note, fibroblasts with a long exposure to IL1β enhance MMPs levels in the lung [28]. MMPs are believed to be involved in several pulmonary diseases. Accordingly, MMP2 expression was found up-regulated in pulmonary fibrosis [29]. Besides the important role of MMPs in extracellular matrix degradation, these molecules are involved in activation of latent TGFβ1 protein [30]. Glassberg et al. have reported a significant increase in MMP2 activity and collagen production in the lung of old female mice [29]. Moreover, Dogru and colleagues have found a dramatic change in lung structure in ovariectomized rats characterized by marked histological abnormality as well as mononuclear cells infiltration, edema, fibrosis and hemorrhage [31]. Moving forward with the previous researches, we found a marked increase in IL1β and MMP2 expression levels as well as perivascular edema and increase of inflammatory cells infiltration in the lung of estrogen-deficient rats.
In recent years, the potential link between inflammation and apoptosis is being actively investigated. There are strong evidence confirming that relation between pulmonary fibrosis and apoptosis. Recent studies have found that TGFβ1 increases expression of apoptotic mediators including Fas-L and caspase3 in the mouse lung [32]. Accordingly, exposure to hyperoxia has also been shown to lead to an increase in TGFβ1 induced apoptosis in the alveolar type II cells [33].
Anti- inflammatory and anti- apoptotic effects of estrogen has been shown in the last decades [1]. It has been suggested that inflammatory and apoptotic biomarkers increase in the lung of post-menopausal animal model [29, 34]. Estrogen replacement therapy (ERT) has been demonstrated to reverse architectural changes in OVX lung [2]. Of note, estrogen treatment has been shown to exert protective effects against pulmonary apoptosis and inflammation in estrogen deficiency animal model [29].
Recent research has shown that estrogen supplementation decreases apoptotic and inflammatory markers including ERK, MMP2 and caspase3 in estrogen- deficient mice [29]. Accordingly, in the present study, estrogen administration remarkably inhibited OVX induced increase in the expression levels of TGFβ1, IL1β, MMP2, ERK, caspase3 in the lung tissue. Moreover, estrogen administration markedly reversed OVX induced histologic inflammation and fibrosis in the lung of estrogen- treated rats.
In the present study, we also aimed to evaluate whether genistein treatment could affect lung fibrosis and inflammation. For the first time this report is describing the effect of genistein treatment on TGFβ1, MMP2, IL1β, Bcl-2, caspase3 and ERK1/2 in the lung of surgical model of menopause. In the present study, genistein administration remarkably down regulated the expression levels of TGFβ1, IL1β, MMP2, ERK1/2 and caspase3 and meaningfully upregulated the expression of Bcl-2 in the lung of estrogen- deficient rats.
Recent finding strongly supports the idea that genistein can reverse perivascular and interstitial lung fibrosis [11]. Genistein has also been reported to exert an anti-inflammatory effect in OVX mice through reducing IL1β serum levels [35]. Of note, genistein is able to suppress IL1β induced MMP2 expression in fibroblast-like synoviocytes of rheumatoid arthritis [36]. Anti- fibrotic effect of genistein has been suggested in the previous studies.
Yuan et al. have reported the effectiveness of genistein treatment in hyperglycemia induced kidney fibrosis by down- regulating of TGFβ1 [37]. It has been reported that genistein protects the hippocampal neurons against apoptosis in OVX rats. Also, long term intervention with genistein leads to a significant decrease in neural apoptosis with upregulation of Bcl-2 in OVX rats [38].
Caspase3 is found to exert a key role in the execution phase of apoptosis [39]. In the present study, we showed that genistein administration markedly ameliorated OVX induced lung injury by decreasing of caspase3 and enhancement of Bcl-2 expression levels, as apoptotic markers, in genistein- treated rats. There has been confirmed many other identified molecules which are involved in cell apoptosis. This finding is simply suggestion for protective effects of genistein and estrogen treatment against OVX induced apoptosis in the lung tissue. Thus, it needs to more evaluation for other apoptotic markers in estrogen- deficient lung.