Effect of Nigella sativa and its bioactive compound on type 2 epithelial to mesenchymal transition: a systematic review

The term epithelial-to-mesenchymal transition (EMT) was first coined by Elizabeth Hay to describe the phenotypical transition of epithelial cells into mesenchymal cells during the primitive streak formation in chick embryo [1]. This transition is a ubiquitous physiological process that plays a major role in healing process and disease progression [2].

EMT resulted in the release of the immotile epithelial cell that was bounded to their basal membrane, into a non-polarized cell that is freely mobile [3]. The transition is further reinforced by increased production of extracellular matrix (ECM) components [4]. At the molecular level, the EMT process is regulated by several molecular events such as transcription factors activation, cytoskeletal reorganization, and specific cell-surface proteins or microRNAs regulation [5].

EMT classification has been made according to their functional consequences; namely type 1, type 2, and type 3 [4]. Type 1 EMT is assigned to the embryological development, implantation and organ development. It is involved primarily during gastrulation and neurulation, whereby the cell transition between the two different state of epithelial and mesenchymal as needed [6]. These cells might be re-induced further as secondary epithelial cells that were involved in organogenesis and development [7]. Type 2 EMT is involved primarily in tissue regeneration [8] and fibrosis [9]. This happens when inflammation initiate the production of ECM by the fibroblast, to help rebuild the damaged tissue site [10]. However, persistent inflammation can lead to tissue or organ fibrosis following incessant wound healing process [11]. Hence, the balance between wound healing and fibrosis via the inflammation process is what defines the type 2 EMT. On the other hand, type 3 EMT, has been specifically assigned for neoplastic cells in regards to their invasive capability seen in their metastasis to secondary site from their primary oncogenic origin [12].

In 2009, a review was made by Michael Zeisberg and Eric G. Neilson regarding biomarkers of EMT [13]. The loss of the epithelial marker, E-cadherin, and the increase expressions of mesenchymal marker, N-cadherin, together known as the cadherin switch, has been nominated as the hallmark of EMT process [14]. The transition into mesenchymal phenotype was also indicated by the elevation of other mesenchymal markers such as fibroblast specific protein-1 (FSP1), discoidin domain receptor tyrosine kinase 2 (DDR2) or fibronectin [15]. Activation of EMT also triggers the production of matrix metalloproteinase [16]. This results in breaking down of the cell-to-cell adhesion that lead to the increased migratory capability of the resulting mesenchymal cell [13].

Following the advancement of molecular biology, a review on the molecular markers of EMT was made by Samy Lamouille, Jian Xu and Rik Derynck in 2014 [5]. They described regulators of EMT at the transcriptional, translational and post-translational level. In terms of transcriptional factors, some important characters are SNAIL, TWIST and ZEB family of transcription factors [5]. Two variants of SNAIL, SNAI1 (Snail) and SNAI2 (Slug) reduce the expression of E-cadherin by repressing its promoter [17]. In cancer cell, activity of TWIST1 has been shown to have dual transcriptional activities, namely the repression of E-cadherin and activation of N-cadherin [18]. Finally, like SNAIL and TWIST, ZEB transcription factors also bind to E-cadherin promoter and function as transcriptional repressors of E-cadherin protein [19].

Nigella sativa or commonly known as black seed or black cumin is one of the most ubiquitous complementary medicine. It is widely grown in the Middle East, Africa and Asia [20]. In Islam, it was mentioned by the prophet Muhammad (PBUH) for its healing properties [21] and currently widely used in the Islamic practice of prophetic medicine [22]. Pre-clinical and clinical studies have shown Nigella sativa to have multiple health benefits such as anti-inflammatory [23], anti-oxidant [24], anti-bacterial [25], anti-angiogenic [26], and organ-protective [27].

On the other hand, a study on Nigella sativa’s chemical composition by supercritical carbon dioxide revealed few bioactive compounds; namely thymoquinone, dithymoquinone, and dihydrothymoquinone. Among these compounds, thymoquinone is the major active compound in Nigella sativa [28]. Thymoquinone by itself exhibit strong anti-inflammatory [23], anti-bacterial [25], anti-diabetic [26] and anti-oxidant properties [29].

Type 2 EMT plays a role in wound healing and tissue fibrosis, which is dependent to the state of inflammation. Inflammation is a complex mechanism that are influenced by bacterial activity [25], inflammatory response [30] and oxidative stress [29]. Balance between the attenuation and sustenance of these factors determine the fate of the tissue. On one hand, wound healing progresses with attenuation of inflammation. Alternatively, tissue fibrosis progresses with the sustenance of inflammation [31]. In this review, a systematic search of the electronic databases was conducted to identify studies done on different events of EMT type 2, including wound healing, tissue inflammation and organ fibrosis treated with Nigella sativa or thymoquinone.

A systematic review of the literature was conducted to identify relevant reports on the effects of Nigella sativa and its bioactive compound on the EMT, particularly in type 2 EMT. A comprehensive search of biomedical science journals in Ebscohost (published between 1823 to August 2019), Ovid (published between 1946 to August 2019) and Scopus (published between 1973 to August 2019) databases was done. The search strategy was adapted from the previous publication looking at the effect of honey in type 2 EMT [32] involving a combination of the following two sets of keywords; (1) thymoquinone OR Nigella sativa OR dithymoquinone OR thymohydroquinone AND (2) epitheli* OR mesenchym* OR transition OR vimentin OR cadherin OR tgf*.

All secondary literatures and any original articles that were not written in English, do not have abstract available and do not fulfil the inclusion criteria mentioned were excluded. Studies focusing on other types of EMT such as type 1 (embryonic development) and type 3 (cancer metastasis) were excluded from the review.

Articles underwent screening process prior to their inclusion in this review. Titles and abstracts were screened first to ensure inclusion and exclusion criteria were adhered. Then, the full text of what remaining were read thoroughly by five independent reviewers to exclude any article that did not meet inclusion criteria. All reviewers must agree on the inclusion of selected articles for the review before the data extraction phase begins. Any differences in opinions were resolved through discussion between the reviewers.

Data extraction was performed independently with the use of a data extraction form. The following data were recorded from the studies: (1) type of experimental model used; (2) treatment groups; (3) summary of the outcomes measured in the study; (4) summary of the study results; and (5) conclusion of the study.

The literature search identified 1393 potentially relevant articles. Four reviewers independently assessed all articles for inclusion and exclusion based on title and abstract. One thousand three hundred thirty-five of these articles were excluded because they were not related to Nigella sativa or its bioactive compound or not related to EMT in wound healing, inflammation, or tissue fibrosis. From the remaining 58 articles, 36 articles were rejected based on exclusion criteria after reading their full text. A total of 22 articles were retrieved for further assessment and data extraction to be included in this review. A flow chart of the selection process, including reasons for exclusion was shown in Fig. 1.

There are 22 articles that were included in the review. All articles were published between the year 2010 and 2018. The studies were categorized according to the type of pathophysiology that they model which were wound healing, inflammation and organ fibrosis.

Nigella sativa was used as oil ointment in five studies [23, 24, 27, 33, 34], and as cream in one study [35]. Thymoquinone was used in 14 studies [25, 26, 29, 36‐46]. Nigella sativa oil and thymoquinone was compared in one study [47]. Since honey made by bee contains the bioactive compound of its nectar origin, a study that investigated the effect of Nigella sativa honey and its propolis was also included in the review [48].

Wound healing of two types of tissue were modelled among the studies included, cutaneous tissue and corneal tissue. In terms of cutaneous tissue, three types of wound model were used, namely incisional wound, burn wound, and diabetic wound. The two studies that investigated the incisional wound physiology uses mouse [25] and rat [35]. For burn wound, three studies using rat were included [29, 34, 48]. Only one study investigated the cutaneous diabetic wound [26]. In terms of corneal wound healing, one study using rat was included [27]. Studies reporting effect of Nigella sativa or thymoquinone on wound healing were listed in Table 1.

In terms of tissue fibrosis, 5 studies that model the tissue fibrosis of different organs were included. For cardiac fibrosis, one study using doxorubicin-induced rats was included [38]. For lung fibrosis, 2 studies with bleomycin-induced rats [23] and paraquat-induced rats [42] were included. For liver fibrosis, one study using carbon tetrachloride-induced rats was included [40]. For renal fibrosis, one study using nitrite-induced rats was included [24]. Studies reporting effect of Nigella sativa or thymoquinone on tissue fibrosis were listed in Table 2.

Progression of wound healing and tissue fibrosis rely on the inflammation state of the tissue [31]. As a result, type 2 EMT researchers focus on the inflammatory properties of the diseased tissue. In terms of inflammation, 11 studies that model inflammation in different organs were included. In terms of the nose inflammation, rhinosinusitis, one study using rats induced with intranasal platelet activation factor was included [46]. Ear inflammation, otitis, is modelled in one study using bacterial infection-induced model [36]. In terms of inflammation following lung injury, 3 studies were included using cardiac ischemia-induced rats [39], cadmium chloride-induced rats [33], and ovalbumin-induced rats [43]. In terms of inflammation in the kidney, 2 studies using diabetic-induced nephropathy model in rats [45, 47] and one study using reperfusion therapy-induced nephropathy in rats [41] were included. One study focussing on liver inflammation using ethanol-induced liver injury model in mice [44] was included. Injury to testicle was modelled in rats in one study [37]. Studies reporting effect of Nigella sativa or thymoquinone on inflammation were listed in Table 3.