This study showed that the hydroalcoholic extract of SS fruit has anti-inflammatory properties on nasal polyp tissue. Increasing GM-CSF improves the inflammatory conditions caused by nasal polyps. On the other hand, our findings showed that the percentage of apoptotic cells compared to control groups in nasal polyp tissues treated with different doses of SE FRUIT extract increased significantly.
NP disease is recognized as one of the most common cases of chronic rhinosinusitis [1, 2]. Currently, functional endoscopic sinus surgery is used for treatment of advanced stages of disease and steroid anti-inflammatory drugs are used for control and treatment at early stages. Surgical treatment is an invasive and high-risk process, and steroids, like many chemical drugs, have various side effects [12, 14]. Although medicinal plants have been introduced as a promising strategy in treatment of some diseases, its therapeutic properties are not fully understood. The current study, we analyzed the effects of SEL. (that is a popular and available herbaceous plant in folk medicine in Iran) on Bad and Bax gene expression, level of IL-5 and GM-CSF cytokines after 24 h exposure to SE FRUIT. Our study specified that using SE FRUIT increase apoptosis in polyp tissue and raise levels of anti-inflammatory cytokines.
It is suggested that multiple mechanisms appear to be involved in increasing inflammation. First, GM-CSF participates in the resolution of inflammation, and IL-3 and IL-5 may also have such properties . In addition, It has been reported that GM-CSF and IL-5 stimulation increase eosinophil gene expression . Similar to IL-5, another cytokines including IL-3 and GM-CSF cytokines are produced as part of the Th2 inflammatory response and are crucial to eosinophil development and function. Second, the physiological effects of each of these cytokines are mainly controlled by the expression of the cytokine-specific receptor, which are highly impact on target cells. Third, lymphoid cells produce GM-CSF during inflammation process and amount of systemic GM-CSF increase dramatically in circulation during inflammation process . We found that SE FRUIT extract reduced the amount of GM-CSF significantly compare to control group (P < 0.0001) and exerts its anti-inflammation property through GM-CSF inhibition.
Our results indicated that SE FRUIT extract increased the level of IL-5 in 50 and 315 µg in vitro, however, the SEs fruits extract decreased the level of IL-5 at 1000 µg/mg SE FRUIT extract in the NP tissues, but it was not significant (P > 0.05). Recently, some studies focused to decrease IL-5 in NP, which is only βc cytokine that have an essential role in differentiation, and proliferation of eosinophils . Van Zele et al. showed that methylprednisolone as a chemical drug exhibited therapeutic effects on NP by decreasing IL-5, eosinophil cationic protein and immunoglobulin E , being consistent with our findings in reducing IL-5 in high doses of the extract. Some studies revealed that low doses of chemical drugs could not induce their therapeutic potency on NP tissues. Besides, allergic diseases that are diagnosed by tissue infiltration of eosinophils can promote long-term tissue injury and fibrosis . Therefore, researchers used the therapeutic strategy such as inhibition of IL-5, and they showed that this can improve esophageal inflammation in children .
Our findings decipher that level of GM-CSF, another key cytokine, significantly decreased after treatment of NP with SE fruits extract. Many studies have demonstrated the importance of GM-CSF and its significant role in the development of NP. Therefore, one of the main goals of researchers is decreasing GM-CSF level and in turn decreases the complications of NP. Taken together, our results indicated that hydroalcoholic extract of SE FRUIT has a significant anti-inflammatory effect on NP through suppression of IL-5 and GM-CSF cytokines. Of noted, SE fruits could suppress GM-CSF factor more effectively rather than IL-5, however, at the high dose the results are similar. Since, the cytokines are directly association with inflammatory cells; their decline can be justified with anti-inflammatory property of SE FRUIT extracts on inflammatory cells, particularly eosinophil.
On the other hands, several studies have indicated that induction of apoptosis in inflammatory cells of the NP, particularly eosinophils, can play a significant role in improving the disease conditions [21, 28]. The results of the GC-MS technique in this study showed that 20.9% of the metanolic extract of SEs contained a natural pentacyclic trisperpenic acid (ursolic acid). Ursolic acid is commonly found in various parts of medicinal plants including leaves, flowers, and fruits and has proven properties such as anti-inflammatory, antioxidant and anti-carcinogenic properties . Ursolic acid has been reported to inhibit the inflammatory factors TNF-α and IL6, LOX-1, VCAM-1, ICAM, IL5, IL13 and IL17 . Another study shows that ursolic acid increases apoptosis in lung cancer cells . In addition, it is also reported that UA via decreasing Bcl-2, Mcl-1, TCTP expression and increasing apoptotic proteins such as TNF-α, Fas, FADD and BAX results in activation of caspase-3 and PARP which induces apoptosis of the liver cancer cells. Therefore, the increase of apoptosis and the decrease of inflammatory factors in this study are probably due to the effect of this substance. Our results of apoptosis detection showed that the percentage of apoptotic cells significantly increased in comparison to the control group in the NP tissues treated with different doses of the SE FRUIT extract in dose dependently. It is suggested that the formation of apoptotic bodies probably inhibit the release of intracellular contents and inactivated inflammatory mediators, therefore exerts its anti-inflammatory property.
Furthermore, high expression of pro-apoptotic genes Bax and Bad confirmed the results obtained from in situ cell death detection with TUNEL. It has been revealed that, the imbalance between Bcl2 and Bax gene expression causes caspase9 activation and leads to apoptosis, indeed, direct activation of caspase3 is another way for causing apoptosis. Our findings decipher that SE fruits extract increased significantly two pro-apoptotic key genes Bax and Bad in NP tissues (P < 0.0001). On the other hand, Beta-sitosterol, one of major component of SEs extract (Table2) has a crucial role in inducing apoptosis in NP tissue. Researchers have been reported Beta-sitosterol as an inducer of apoptosis through the activation of P53 gene and induction of cell cycle arrests may have contributed to increased apoptosis in NP tissue after receiving SEs fruit extracts. Several investigations on drug treatment of NP have shown that inducing transduction of pro-apoptotic genes increases the expression of these genes in NP, leading to apoptosis induction in inflammatory cells, and as a result, the treatment of NP .