# Application of Conditioned Medium for In Vitro Modeling and Repair of Respiratory Tissue
## 4 Discussion
Consequently, it is crucial to thoroughly evaluate the origin of CM and its potential consequences prior to employing it for therapeutic purposes. The dissimilarities observed in the impact of CM from respiratory and non-respiratory tissues could be attributed to variations in the molecular makeup of the CM and the distinct physiological characteristics of the cells responsible for its secretion [70]. The molecular mechanisms and key protein factors involved in the therapeutic actions of the secretome have received attention. Nevertheless, they have not been fully explored, despite the importance of secretome research, which requires wide characterization before clinical research can be conducted. In some in vitro studies related to secretome applications from respiratory diseases, IL-6 expression became the protein of focus rather than H2 AXy, p21, TNF-\(\alpha\), and IL-8. This was because the secretome from unhealthy tissue was induced to the epithelium and altered IL-6 production, which is involved in activating EMT and metastasis [45; 48; 49; 50]. IL-6 stimulates the IL-6 receptor (IL-6R), which initiates signaling via the Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling pathway and the NF-\(\kappa\)B signaling pathway [71], hence triggering an inflammatory reaction and activating EMT processes. Figure 2 provides a detailed overview of the mechanism of action of IL-6. An in vitro study reported EGFR activation induced by activation of the ERK1/2 signaling cascade present in the secretome, which aided wound healing to the epithelium [57]. Another study reported that AKT and PI3K signaling were involved in secretome application in vitro, as they induced a Na\({}^{+}\) influx through the epithelium sodium channel (ENaC) activity [59]. Contrastingly, only one in vitro study used a bacterial secretome (_S. aureus_), which involved ZO-1 expression [64]. The ZO-1 protein aids cell adhesion and TJ management; hence, the secretome created ZO-1 expression discontinuity and damaged the respiratory epithelial TJ [64]. IL-8 expression was the focus in terms of particle-CM, as the IL-8 protein was increased due to the cytotoxic effect of the proinflammatory cytokines when the epithelium was exposed to the CM [63]. A comprehensive summary of the findings in this study is presented in Figure 3.  
Figure 2: Depicts the role of IL-6 in activating the JAK-STAT pathway, which is responsible for many of the observed therapeutic effects of CM on respiratory tissues [72]. IL-6 binding to its receptor induces the activation of the JAK-STAT signaling pathway, which, in turn, leads to the phosphorylation of STAT proteins and their subsequent dimerization, nuclear translocation, and regulation of gene transcription, including inflammatory cytokines. Data from [PERSON] et al. (2022) [72]. Recent research has also demonstrated the use of exosomes rather than secretomes for in vitro applications. Although both secretions have received attention for their therapeutic potential in respiratory regeneration, they focus on two separate aspects. While the secretome mainly consists of messenger chemicals discharged to the outside, including microvesicles and exosomes, both secretomes and exosomes confer distinct benefits and target various metabolic pathways and affect the respiratory epithelium. The secretome was occasionally linked to the immunological and endocrine system pathways and protein processing in the endoplasmic reticulum. Contrastingly, exosome-specific proteins were mostly related to endocytosis, cell junctions, platelet activation, and other cell signaling pathways [73]. In contrast to the highly complex nature of the secretome, exosomes can be engineered to carry a specific cargo based on their selectivity for parent cell types [74], enabling targeted delivery to specific cells or tissues and leading to improved therapeutic outcomes with a greater specificity and reduced risk of adverse effects, including potential protumor effects [75; 76].