## 3 Results and Discussions
### Evaluation of Biomolecular Markers by Immunoblotting  
Immunoblot analysis was carried out on HS68 cells in order to evaluate the variations of two proteins related to oxidative stress, in response to the treatment with _M. nodiform_ SFE extract (AOX) and hydrogen peroxide (HP). The results are shown in Figure 5.  
Integrins are heterodimeric transmembrane glycoproteins that participate in different cell processes, such as cell differentiation, migration, survival and proliferation [19; 53; 54]. In fibroblasts, \(\alpha\)1 is known to activate the RAS/ERK proliferative pathway and has a pro-invasive function in certain cancers and it is generally considered as a stress marker [19]. In our experiment, the protein integrin \(\alpha\)-1 significantly increased, respect to the control, (p \(<\) 0.05), in cells induced to oxidative stress by HP, without antioxidant protection. On the contrary, cells of the treatment AOX/HP, that were pretreated with SFE extract, prior to the induction of oxidative stress by hydrogen peroxide (HP), showed a reduction of its levels, compared to the control, highlighting the protective effect of the extract against oxidative stress. Integrin \(\alpha\)1\(\beta\)1 is involved in the process of remodeling the extracellular matrix and reduces the expression of the type I collagen gene, which is the most abundant component of connective tissue. Matrix remodeling is an important event, characterized by the deposition of new collagen, a process that is activated for normal organogenesis, wound healing, but also during the invasion of  
Figure 5: Results of the immunoblotlot of Integrin \(\alpha\)1 and Sirt 1 in HS68 cells. Control: cells maintained in standard culture conditions; HP: cells exposed to hydrogen peroxide (10 \(\upmu\)M); AOX/HP: cells treated with 10 \(\upmu\)g/mL of _M. nodiform_ SFE and then exposed to HP (10 \(\upmu\)M). \(\beta\) actin was used as internal control. The immunoblotlot image, in (**c**), is one representative of the three separate experiments. The related protein quantifications are represented in the graphics (\({}^{\circ}\)_p_\(<\) 0.05 vs. control): (**a**) relative density of Integrin \(\alpha\)1 protein; (**b**) relative density of Sirt 1 protein. metastasis [55]. Cellular stress influences matrix remodeling, deregulating the biological pathways in which integrins are involved [56]. Other studies reported the increased expression of integrins in a stress situation, such as in mouse skin exposed to UV, that showed the expression of integrins \(\alpha\)2, \(\alpha\)5 and \(\alpha\)1 [56], or in human endothelial cells of the umbilical vein, in which the expression of the \(\alpha\) 1 subunit resulted induced by factors such as Tumor necrosis factor alpha (TNF-\(\alpha\)) and retinoic acid [57]. Sirtuins are a family of nicotine adenine dinucleotide (NAD) dependent protein deacetylases that regulate cellular function through deacetylation of a wide range of target proteins [20] and are also involved in stress resistance, apoptosis, senescence, aging and inflammation. They regulate several physiological and pathological events, including neurodegeneration, age-related disorders, obesity, heart disease, inflammation and cancer [58]. Our results showed that, independently from the treatment with _M. nodiforum_ SFE extract, the protein Sirt 1 evidenced a significant decrease (p \(<\) 0.05) after the exposition to HP, respect to the control, indicating its sensitivity to situation related to oxidative stress. In particular, sirutin 1 has anti-inflammatory, anticancerous effects and regulates growth modulation and cellular metabolism function [59]. SIRT1 regulates inflammation mediators, and its activation inhibits the production of factors such as TNF-\(\alpha\), MCP-1 and IL-8. The suppression of pro-inflammatory cytokine production by SIRT1, is related to its negative regulation of NF-kB activity by deacetylation of the RelA/p65 lysine 310 subunit [59].