# Evaluation of In Vitro Antihypertensive and Anti-Inflammatory Properties of Dairy By-Products
## 3 Results and Discussion
### Effect of D-P3 on Expression of Inflammation-Related Genes  
Monocytes differentiate into macrophages, following polarization depending on a variety of stimuli, and diverse inflammatory pathways are activated. In response to different microenvironment, various functional phenotypes are detected [64]. Macrophages secrete inflammatory cytokines, including IL-1\(\beta\) and IL-6, as well as TNF-\(\alpha\). In addition, certain enzymes, such as iNOS and COX-2, play an important role in the development of inflammation-related diseases. In this study, the relative expression of _Nos2_, _Ptgs2_, _II6_, and _Tuf_ inflammation-related genes was quantified in response to treatment with D-P3 in LPS-stimulated RAW 264.7 macrophages. As shown in Figure 5a, _Nos2_ expression was found to be higher (\(p<0.05\)) in BL-D-P3 when compared with caprine and ovine SW-D-P3 but not with bovine and mixed ones. Also, higher _Nos2_ mRNA levels were observed for bovine SW-D-P3 compared to caprine and ovine SW-D-P3. On the other hand, no statistically significant differences were observed between SW treatments and BL-D-P3 regarding _Ptgs2_, _Tuf_, and _I16_ expression (\(p>0.05\); Figure 5b, c, and d, respectively). Figure 6 shows the quantification of transcription levels of genes relevant to the immune response for YAW fractions. No statistically significant differences were observed between the YAW-D-P3 samples either between different animal origins or with BL-D-P3-treated cells regarding _Nos2_, _Ptgs2_, _Tuf_, and _I16_ expression (\(p>0.05\)). Figure 5: Effect of SW-D-P3 on LPS-induced mRNA expression in RAW 264.7 cells. RAW 264.7 cells were treated as follows: LPS, cells stimulated only with 1 \(\mu\)g/mL LPS; D-P3, cells stimulated with LPS and subsequently incubated in the presence of SW-D-P3 (caprine, ovine, bovine, and mix; 0.038% \(w/v\) refers to starting SW-D protein) or BL-D-P3 (blank) for 24 h. The expression levels of (**a**) _Nos2_, (**b**) _Ptgs2_, (**c**) _Tnf_, and (**d**) _ll6_ were measured using qPCR and were normalized to housekeeping genes (Actb and Cyc1). Data are presented as mean \(\pm\) SEM of two independent experiments. Columns with different letters within the same panel are significantly different (\(p<0.05\)); ns = not significant (\(p>0.05\)). Previous studies reported that natural non-dairy compounds, including protein and peptides, have anti-inflammatory activity by suppressing _Nos2_ expression, resulting in the inhibition of NO production, as well as by down-regulating pro-inflammatory gene expression, most of the time, in a dose-dependent manner [65; 66; 67; 68; 69]. The nuclear factor-kappa light-chain-enhancer of activated B cells (NF-kB) pathway is a crucial signaling pathway responsible for maintaining the defense system against inflammatory stimuli, either endogenous or exogenous (like LPS). The production of cytokines (IL-1\(\beta\), IL-6, and TNF-\(\alpha\)), the induction of iNOS, and the expression of COX-2 are regulated by the participants in the NF-kB canonical pathway [66]. [PERSON] et al. reported that [70] two buffalo whey peptides significantly inhibited the expression of inflammatory mediators, _iNOS_, _TNF-\(\alpha\)_, and _IL-6_, in stimulated with 1 ug/mL LPS murine RAW 264.7 macrophages, in a dose-dependent manner (50-200 ug/mL), indicating the protective mechanism of these peptides against cell inflammation damage. Another study indicated that the <1 kDa fraction from WPH, as well as two subsequent sub-fractions, showed inhibitory activity on IL-1\(\beta\), COX-2, and TNF-\(\alpha\) mRNA expression in LPS-induced RAW 264.7 cells [71]. According to a previous study, the addition of fermented camel milk (at concentration of 0.25 mg/mL) resulted in the inhibition of pro-inflammatory cytokine production of TNF-\(\alpha\), IL-6, and IL-1\(\beta\) in LPS-stimulated murine macrophages [72]. Another study reported the  
Figure 6: Effect of YAW-D-P3 on LPS-induced mRNA expression in RAW 264.7 cells.