Laying hen has been known as a robust model of spontaneous ovarian cancer in human due to the expression of some molecular markers, pro-inflammatory mediators, site of origin (oviduct and ovary), performed at relatively little cost and same presentation and progression to the human disease that appears to be associated with prolific ovulation and aging [7, 16]. Moreover, similar to women ovarian cancer, there are comparatively more immune cells in laying hens’ ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors [16, 17].
In the majority number of evidence, the stimulation of beta-2 adrenergic signaling was mentioned to involve in multiple cellular processes that contribute to the initiation, progression, and metastasis, including inflammation [18, 19]. Nonetheless, it has proven that in the tumor microenvironment, which is largely orchestrated by inflammatory cells [20], the beta-2 adrenergic transmembrane signaling is impaired and the number of β2ADR reduces due to increasing the number of deficient receptors or cell types lacking receptors, in some cancer types [21]. Therefore, the GC and BAA combination could be as an efficient strategy to restore the transmembrane β2ADR number and its cellular signaling. Therefore, this could be as the first report to demonstrate the effects of the synchronous supplementation of GC and BAA in the ovarian inflammatory cells on laying hen model of ovarian cancer.
Ovarian mRNA expressions
As shown in the Fig.1-A, compare to control, we observed the significant down-regulation of β2ADR in Individual treatments of BAA, GC, BB and GC + BAA1, 2, and significant up-regulation in GC+BAA3 of β2ADR mRNA expression. Nevertheless, in comparison with BAA and GC, all of the combination groups indicated a significant increase in β2ADR mRNA expression that are in agreement with previous reports regarding increase in mRNA expression of this adrenergic receptor [13, 15].
Cellular density of β2ADR derives the various factors like the type of tissue [22, 23], cellular age [24], inflammatory condition [23], and overstimulation of β2ADR in the face of excessive BAA exposure [22]. Concerning the factors mentioned above, a decrease in β2ADR mRNA expression could probably be as a consequence of β2ADR overstimulation BAA Salmeterol (1 ppm BW) and prolonged agonist exposure (four weeks) in the hens’ ovarian epithelial tissue within the inflammatory condition of ovulation. On the other hand, according to the results derived by the previous studies about the evaluation of BAA Salmeterol on membrane β2ADR density [25, 26], it has been proven that the administration of BAA Salmeterol brings about a considerable stabilization of membrane β2ADR because of having a very low efficacy for stimulating arrestin-2 and G protein-coupled receptor kinase enzyme (GRK) phosphorylation as essential mediators to induce β2ADR desensitization, down-regulation and internalization. Improvement of membrane β2ADR density could be as an effective factor to downward cellular synthesis and degradability turnover of β2ADR proteins and consequently causes to decline its gene transcription. Despite this fact that GC gave rise to up-regulate β2ADR mRNA expression in some of the past researches, but the studies like Tan et al. 1997 reported high-dose inhaled GC Fluticasone did not up-regulate lymphocyte β2-AR as compared with a single dose of oral prednisolone [27]. In this regard, similar to BAA Salmeterol, GC Fluticasone has been indicated to decrease in protein expression of arrestin-2 and GRK [28, 29] and as a result, causes to improve membrane β2ADR density and decline its cellular mRNA. Our results demonstrated that, compare with the other groups, the combination groups had the different behaviors, so that GC + BAA1 and 2 down-regulated and GC + βBAA3 unregulated β2ADR mRNA expression as compared to control; nevertheless, all of the combination groups enhanced this expression In comparison to BAA Salmeterol and GC Fluticasone that were in accordance with the some documents [13, 30]. They mentioned BAA and GC combination may regulate β2ADR function by increasing both of protein and mRNA expression of the receptor, restoring G-protein/ β2ADR coupling, and inhibiting β2ADR down-regulation given rise after exposure to high doses of BAA on respiratory disorders. Thus, it’s supposed that compare to individual usage of BAA and GC, supplementing the hens with all of combination groups caused to up-regulate β2ADR mRNA expression via the positive effect of GC on the mRNA expression of this receptor, as mentioned above. β2ADR has been shown to play a considerable role in different ovarian events like ovulation, hormonal secretion, and puberty [31-33]. However, it has been reported that this receptor exhibited significant overexpression in tumor tissue on reproductive malignancies like ovarian cancer [34]; it has been recommended that beta blocker usage reduced the chance of death compared with that of non-users [34, 35]. Anyway, on the one hand, according to this fact that reduction in egg-laying frequency (ovulation rate) results in a decrease in ovarian cancer [36, 37], on the other hand, hens treated by all of the combination groups had a much less ovulation rate than control, BAA (BAA elevated significantly compare to control) and BB (Table 3), it could reach to this opinion that increase in membrane density of β2ADR possibly doesn’t boost the beginning and severity of ovarian cancer in laying hens as the model of women’s ovarian cancer. This opinion was stood out when accompanied with the recent documents indicating that there are no associations between BB usage and cancer survival, prognosis, and mortality [38-40].
As the rate-limiting enzymes, cyclooxygenase-1 and 2 (COX) have the critical role in the various physiological roles, and be involved in different ovarian reproduction processes like ovulation [41]. Although, COX-1 has been known to be expressed in most cells and tissues and remains in constant expression under most physiologic conditions, COX-2 is inducible and generally only expressed in response to various inflammatory reactions [42]. However, both of COX-1 and 2 have been reported to be up-regulated throughout the ovarian carcinoma [16]. Here, we reported that the supplementation of BAA Salmeterol and BB propranolol down-regulated both COX-1 and 2, and GC Fluticasone resulted in the significant up-regulation of COX-2 and down-regulation COX-1. According to Shore’s study, TNF-α and IL-1β synergistically perform to promote β2ADR desensitization through the induction of COX-2 expression [43]. Therefore it’s believed that the down-regulated TNF-α and IL-1β derived by the supplementation of BAA Salmeterol (Figure 2, A and D) caused not only to down-regulate COX-2 expression but also lead to a decrease in β2ADR desensitization. Our COX-1 result was in agreement with some reports that have demonstrated that glucocorticoids down-regulate COX-1 gene expression [44]; nonetheless, dislike our COX-2 result, several documents indicated that COX-2 expression is inhibited by glucocorticoids [45, 46]. However, some studies reported that glucocorticoid therapy enhances COX-2 expression. In this regard, Sun et al. 2008 indicated that GC induced COX-2 gene expression via inducing the interaction of glucocorticoid receptor with C/EBP-β (CCAAT/enhancer-binding protein-β) and activation of p38 MAPK (mitogen-activated protein kinase) in cardiomyocytes; in fact, activation of glucocorticoids and their receptor were necessary for COX-2 gene expression due to the binding of both glucocorticoid receptor and C/EBP-β to COX-2 promoter [47]. Although, all of the combination groups didn’t have the more anti-inflammatory efficiency for down-regulating COX-2 mRNA expression than BAA group, GC + BAA1 and 2 had the less COX-2 mRNA density compare to the control and GC group. However, except for the effect of GC + BAA2 on COX-1 compare to control, the other combination groups up-regulated COX-1 mRNA expression as compared to control, BAA, GC, and BB groups. Therefore, according to decreasing COX-2 observed from combination groups, some of these groups are possibly capable of having the anti-inflammatory role via down-regulating COX-2.
Cytokines as products of immune system have also been proven to be synthesized by an extensive range of non-immune cells, like the normal ovarian cells; and their action in the ovary has been described as the motivational processes of follicular development, activation of leukocytes required for ovulation, and tissue remodeling during ovulation [48]. Meanwhile, cytokines are reported to associate with ovarian cancer via regulating growth, signaling, and differentiation of both tumor and stromal cells and may affect on behavioral traits of malignant cells [49]. Among these, TNF-α, IL-1β and IL-6 [49] as the pro-inflammatory cytokines, and IL-10 as an anti-inflammatory cytokine [50] have been shown respectively to promote and restrict ovarian tumor genesis, growth, and progression. According to the diagrams indicated in Fig. 2, except IL-1β, the other mentioned cytokines showed the similar changes in of mRNA expression when compared to control; so that, all of the treatments either individuals or combination groups have shown the less IL-1β mRNA expression than control; while, GC + BAA3 group had the highest mRNA expression of TNF-α, IL-10, and IL-6 among the others. However, in all of the mentioned cytokines, the individual groups of BAA, GC and BB had the lowest mRNA expression when compared to control and combination groups. Despite this fact that some studies reported that other BAAs led to a significant increase in mRNA and protein expressions of three cytokines TNF-α, IL-1β, and IL-6 [51, 52], Salmeterol has been known to inhibit the secretion of these cytokines . In this regard, Hu et al. showed that Salmeterol inhibits the activation of MAPK and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ƙB) [53] as main pathways of induction of some pro-inflammatory cytokines including IL-1β, IL-6 and TNF-α [54]. Anyway, the lower mRNA expression of IL-10 in the BAA group was in contrast to the previous studies that have worked not only on Salmeterol [55, 56] but also on the other BAAs [57]. Similar to BAA, our results agreed with other researches having documented that GC markedly suppressed mRNA expression of key pro-inflammatory mediators including TNF-a, IL-1β, and IL-6 [58, 59], and was unlike to others that had mentioned GCs may regulate inflammatory action by increasing IL-10 mRNA expression as well as higher serum IL-10 concentration [60]. Although having the higher mRNA expressions of mentioned cytokines than individual groups, some combination groups down-regulated the cytokines mRNA expression compare to control. In agreement with our results, some researches have proven that the combination of these drugs induce a significant decrease of IL-1β, IL-6, and TNF-α [61, 62]. In this regard, Fragaki et al. 2006 suggested that combination of Salmeterol and Fluticasone is capable of decreasing pro-inflammatory cytokines by reducing to reduce the NF-ƙB and Activator protein 1 (AP-1) activity via 1) the reduced activation of extracellular signal-regulated kinase (ERK)1/ERK2 and c-Jun N-terminal kinases (JNKs) pathway and 2) the reduced phosphorylated form of IƙBα leading to NF-ƙB nucleus binding inhibition [62]. Likewise, IL-10 was reported to increase via administrating GC and BAA more effective than the sole administration of the GC [63] that in our results was just similar to GC + BAA3 group as compared to control; nevertheless, all of the combination groups had the higher mRNA expression of IL-10 than the individual groups.
The function of ovarian hormones
As major reproductive steroid hormones, estradiol, progesterone, and testosterone (androgen) have been confirmed to play the functional roles to regulate growth, differentiation, and function of an extensive range of target tissues in the females’ reproductive system [64]. It has been reported that there is an association between ovarian cancer and ovarian hormones [65]; on the one hand, Estrogen was shown to regulate growth and differentiation in the normal ovaries and has mutagenic effects. Beside, some evidence confirmed that estrogen accelerates the inflammatory process through the up-regulation of cytokines including IL-1β, IL-6, TNF-α and matrix metalloproteases (MMPs) [66]. Progesterone, on the other hand, has been indicated to induce apoptosis and reduce cell membrane permeability that gives rise to decrease invasive potential [67]. This storied hormone has been reported to have a protective role in preventing inflammation during pregnancy by reducing of IL-6 and TNF-α, and by the recovery of antioxidant enzyme performance in some tissues [68]. Likewise, Emerging evidence has mentioned that androgen-depended pathways have also been known to plays an important role in the pathogenesis and progression of malignancies, prostate, breast, and ovarian cancers [69, 70]. More interesting that androgen therapy offsets the inflammatory process and reduces the intensity of disease by mechanisms inhibiting inflammatory cytokines expression and function like TNF-α, IL-1β, and IL-6 [71]. About the results expressed in Fig. 3 A-C, the birds supplemented by BAA and BB, had significantly higher serum estradiol content that was in contrast to the GC and combination groups showing the lower estradiol content, as compared to control. Similar to our results in estradiol, serum progesterone content was the highest and lowest in the BAA and GC groups, respectively and all of the combination groups and BB statistically showed similar serum progesterone content compare to control. Except for BAA and GC+BAA3 groups, the other treatments also indicated the same behavior on the serum androgen. In keeping with our results, the previous evidence reported that catecholamines elevated serum estradiol, progesterone, and androgen concentrations in the experimental animals [72-74]. This up-regulation has been mentioned that not only has directly been derived by theca layer stimulation of the ovarian follicles [72] but also has been influenced via indirect regulation of the pituitary gonadotropes response to GnRH [75] that these routes are activated through current beta-2 adrenergic signaling of formation of cAMP. According to the obtained finding of previous studies, reduced content of serum estradiol and progesterone observed on the GC group in our study could be derived by the factors like inhibition of hypothalamus- Pituitary –gonads [76], inhibition of estradiol activity by increasing the expression sulfotransferase [77], and decreasing luteinizing hormone (LH) receptor number [78]. Because the administration of BAA+ GC has been defined as an anti-inflammatory strategy in respiratory disorders, the valid evidence was not found to document ovarian steroids hormones change in experimental animals’ plasma treated by combination strategy of BAA and GC. For this reason, the presentation of our results about plasma ovarian hormones changes could be defined as the first report that mentions the effect of combination groups on these hormones.
The function of cellular and humoral immunities
Because of incessant ovulation and chronic inflammation, the immune system is assumed to be defined as the main mediator of ovarian cancer [79]. In this regard, the increasing documents are indicating that systemic inflammatory activation derived by cancer cells anticipates tumor progression via inducing cancer proliferation and metastasis or promoting angiogenesis [80, 81]. Therefore, the inflammatory markers, like the neutrophil (called as heterophile in the birds)-lymphocyte ratio (NLR) has been mentioned as a considerable index of the systemic inflammatory response for predicting the prognosis of different cancers like ovarian cancer [81]. Generally, the factors that increase ovarian cancer risk were accompanied to higher NLR, and factors decreasing risk were associated lower NLR [82]. The elevation of NLR derived ovarian cancer, is created via increasing circulating neutrophils and decreasing lymphocytes counts. Our results showed in Fig. 5 (A-C) that the administration of GC Fluticasone gave rise to significantly boost neutrophil and NLR and decrease in lymphocyte as compared to control and BAA groups. In agreement with our results, some of documents have reported that on the one hand, GC not only causes to elevate the accumulation and survival of neutrophils [83] but also to up-regulate of anti-apoptotic Bcl-2 (B-cell lymphoma- 2) family members, activate NF-κB, Suppress components of the extrinsic pathway of apoptosis, and induce signaling molecules such as MAPK phosphatase-1 (MKP-1) and Serum and glucocorticoid activated kinase-1 (SGK-1) [84] that promote inflammatory aspects in neutrophils. On the other hand, GCs indicated the different behavior on lymphocyte numbers rather than neutrophil because GC results in the skew of T cells, activation of NF-κB via stimulation of Toll-like receptors [85], and activation of death-inducing genes and consequently induce apoptosis [86]. Observed increase and decrease in neutrophils and lymphocyte respectively resulted in a rise of NLR in hens administrated by GC Fluticasone that was similar to the some studies showing that treatments using GC had higher NLR values that are mainly derived by higher neutrophil counts [87]. Despite being none significant as compared to control, the BAA group had the lowest neutrophil percentage and NLR among other treatments and except to GC, BAA had the same effect on lymphocyte percentage with the other treatments. In this regard, some documents have proved that activation of the β2ADR inhibits inflammatory responses in neutrophils via the various intra-cellular pathways like clearance of cytosolic Ca2+ and inhibition of the generation of superoxide anion (O2(•-)) production [88, 89] and release of acetylcholine that exerts its anti-inflammatory effects binding to alpha-7 nicotinic receptors [90]. Reduced neutrophil percentage observed in the BAA group could give rise to a significant reduction in NLR compare to GC and GC + BAA1. The nearest to these results, some studies have expressed that NLR could be the independent and straightforward predictor for inflammation-induced-respiratory disorders like asthma and COPD [91] that are improved by BAA [12] and combination of GC and BAA [15]. Despite having a significant increase in GC + BAA1, neutrophil percentage was statistically similar on GC + BAA2 and 3 as compared to control. Although having more neutrophils count than the BAA group, all of the combination groups indicated the anti-inflammatory effect due to having less neutrophil count compare to the GC group. In keeping with our results, some researches announced a reduction in neutrophils percentage for the patients administrated by Salmeterol plus Fluticasone propionate in the inflammation related respiratory disorders [92]. As result of the decrease in neutrophils percentage in combination groups, NLR was reduced in these groups as compared to the GC group; however, except for GC + BAA1, GC + BAA2, and 3 statistically had the similar influence on NLR compare to BAA, BB and control.
As easily extracted from serum via minimally invasive blood collection, autoantibodies or natural antibodies have become of particular interest as cancer biomarkers for current decades [93]. They have been confirmed to show the elevated concentration in very early cancer stages and during the transition to malignancy [94] and have been proven in patients with several carcinomas like ovarian cancer [95]. According to the main autoantibodies, IgG and IgM subclasses have been known to perform as the most and milder sensitive immunological responses respectively concerning protein-specific glycosylation profiles in serum of EOC patients as compared to healthy individuals [96]. Moreover, there are different documents regarding IgG and IgM effects on the growth of tumor cells. While Staff et al. 2012 mentioned that IgM might have an anti-tumor role in addition to IgG [97], Manjula et al. 1992 observed an Elevated level of serum IgG or IgM antibodies is in patients with cancers of epithelial origin, like breast, colon, and liver cancers [98]. However, Qiu et al. 2003 achieved to this fact that tumor-derived IgG was involved at least, in part, in the survival and growth of epithelial tumor cells because anti-human IgG induced apoptosis and growth inhibition of cancer cells in vitro and in vivo [99]. According to the results shown in Fig. 5, the birds administrated by GC had the least whole Ig, IgG, and IgM among other groups that were in agreement with evidence proved that corticosteroids appear to have a negative correlation on levels of some serum immunoglobulins [100]. Regarding this effect, GC has been reported to decrease B cells through promoting intracellular pathways of apoptosis and death-inducing genes [86], modulating peripheral B cell maturity via inhibiting activation-induced cytidine deaminase (AICDA) expression [101], dephosphorylation of ERK‐1/2 via increased dual‐specificity protein phosphatase1 (DUSP1) expression [102], and down-regulating Bruton Tyrosine Kinase (BTK) for B-cell activation [103]. Except for a significant enhancement of IgM, it was not observed the considerable difference in whole Ig and IgG in the BAA group compare to the control. Nevertheless, it is supposed that the comparative advantage of Ig production in some of the combination groups could be derived by enhancing of beta-2 adrenergic signaling- mediated pathways in these groups due to more beta-2 adrenergic receptor in B- cell membrane; so that Sanders 2012 mentioned activation of two pathways, namely LynCD19-Akt-NF-kB p50-p65 and PLCγ0032α-PKC-p65 having the role of an increase in the amount of IgG1 secreted per B cell, were found to converge by CREB that is emerged by beta-2 adrenergic signaling- mediated pathways [104]. In contrast to our thinking, Lee et al. 2016 reported a significant down‐regulation for both B cell proliferation and IgG expression (both mRNA and protein) using Fluticasone : Salmeterol combination with 1:10 ratio and Fluticasone alone [102].
Ovarian and body functions
Ovulation is defined the major contributor to emerge ovarian cancer that was supported by two hypotheses of incessant ovulation (Fathalla’s incessant ovulation hypothesis) and inflammation [37]. Fathalla has been theorized the continuous involvement of the ovarian surface in the process of ovulation because of repeated processes of rupture and repairing the wound of the ovarian surface. Over time, these processes boost the possibility of errors occurring during DNA replication. On the other hand, according to the inflammation hypothesis, the ovulation-related events have been reported to resemble an inflammatory reaction that accompany with leukocytes infiltration and production of inflammatory mediators like cytokines, Vascular growth factors (VEGF), Prostaglandins, and intracellular signaling pathways closely associated with inflammatory reaction [105]. Concerning these hypotheses, it is supposed that the inhibition of ovarian cancer will occur if the process of ovulation is modified by suppressing inflammatory mediators and signals. Regarding the results presented in Table 3, the laying hens supplemented by BAA Salmeterol significantly showed a more ovulation rate and being in contrast with GC and combination groups that had a lower egg-laying frequency as compared to control. Moreover, the GC group showed the smallest pre-ovulatory follicle (F1) size among the other groups, insofar as, the follicles of F2 to F5 were not observed (N.O.) in the GC group. However, BAA, BB, and GC+ BAA 3 statistically indicated the similar follicle size F1 and except GC, all of the treatments had a similar size at follicles F2 to F5 compare to the control. In addition to the influence of inflammatory events, the factors like nutritional and metabolic factors and relating hormones of the hypothalamus-pituitary-ovary axis have been documented to play the fundamental roles in the function of ovulation and follicular development and differentiation. About the effect of nutrition and metabolic status, some evidence demonstrated energy balance, nutrients (fatty acids, glucose, and amino acids), and metabolic hormones like insulin, IGF-I and growth hormone implicate in ovarian functions such as the follicular development and ovulation [106]. GnRH, gonadotropins, and ovarian hormones, on the other hand, have been reported to preliminary effects on follicular development and ovulation [107].
According to our gained results on mRNA expression of ovarian inflammatory mediators and ovarian hormones, and nutrition and metabolic status explained above, it could reach to this belief that each treatment influenced on ovulation rate and follicular size by the effect on the situation of ovarian hormones and metabolic status. Regarding the results shown in Fig. 6, food consummation was elevated in the BAA group and decreased in GC and combination groups that were in agreement with previous study [108] in the GC group. Enhancement of food intake observed in the BAA group gave rise not only to improve live BW that represents positive energy balance but also to be as one of the confirmed reasons [106] for increasing ovulation rate and follicular development. Moreover, BAA was shown to increase insulin and IGF-I [109, 110] and growth hormone [111] that promote ovulation and follicular growth. Enhanced follicular growth and ovulation rate could also be as the results of elevated serum estradiol and progesterone in the birds administrated by BAA Salmeterol. As one of the contributing factors of ovulation, inflammatory mediators that their mRNA expressions were down-regulated in the BAA Group, do not seem to have enough influence on ovulation and follicular growth in this group compare to ovarian hormones and metabolic status. Reduced ovulation rate and follicular growth observed in the GC group could be derived by: 1) Decrease in food intake that resulted in negative energy balance and consequently loss of live BW observed in the GC group, 2) induction of Insulin resistance [112], disturbance of IGF-I [113] and down-regulation of growth hormone [114], 3) observed down-regulation of estradiol and progesterone, and 4) observed reduction in mRNA expression of inflammatory mediators. Despite indicating the statistically similar size at the most classes of follicular growth, like the GC group, combinations groups had lower ovulation rate than the control, BAA, and BB . In this regard, authors believe that BAA and GC could have the dominant and synergic actions on some effective factors on ovulation intensity and follicular growth. BAA is supposed to suppress the inhibiting role of GC on some of the reproductive hormones and metabolic mediators like insulin, IGFs and growth hormone; while as the synergic effect, BAA promotes GC effect on the decrease in inflammatory mediators and finally this BAA and GC interaction gives rise to improve the follicular size and decrease in ovulation rate in combination groups.