## 4 Discussion
### Strengths and Limitations  
The present study is associated with several strengths. The present experiments are the first pathway studies of the tRAS for IVD cells. As we used human NP cells from patients' IVDs in this study, these findings could directly impact clinical and therapeutic strategies in contrast to preclinical studies in other species that often need to be verified in humans first. We provided novel evidence that treatment with losartan suppresses pro-inflammatory and degenerative responses to inflammatory stimuli in human NP cells. We further revealed an interaction of the tRAS with the PPAR\(\gamma\) pathway, which could be of potential interest for future pathway studies. Some limitations need to be addressed to interpret the results adequately. Our studies were conducted in vitro using a 2-dimensional monolayer cell culture. In vivo interactions cannot be perfectly simulated with this methodological approach and the concentrationsused for the intervention arms. The concentration range of losartan chosen in this experiment was based on previous in vitro pathway studies with other human cell types as evidence for intervertebral disc cells is scarce [69; 76; 77]. Therefore, the concentrations used in our cell cultures might not reflect the available tissue concentrations in humans after usual orally available doses of losartan. Reported oral doses of losartan range between 25 and 200 mg per day. Its terminal half-life is approximately 2 h, but its active metabolite EXP3174, which has a much more inhibitory effect on AGTR1 (up to 40-fold more potent), can reach a half-life of 6-9 h [78]. Reported maximal plasma concentrations in humans reached 84.5 ng/mL after a single oral administration of 25 mg and 1394.9 ng/mL after single oral administration of 200 mg losartan. For its active metabolite, these maximal plasma concentrations were 188.9 ng/mL and 2219.0 ng/mL, respectively [79]. This would suggest that oral doses cannot accumulate the tissue concentrations needed for the anti-inflammatory effects seen in our results, at least in vitro. New drug delivery and administration techniques will be needed to reach these tissue concentrations. Another approach would be to find more potent inhibitors of the pathological tRAS arm or to evaluate stimulation of the protective tRAS arm components, which possibly will lead to anti-inflammatory effects in much lower concentrations. However, the present preclinical experiments help us understand the role of the tRAS and its inhibition in human NP cells, and support future planning of potential ex-vivo organ culture models followed by the in vivo studies needed in order to confirm these results. Furthermore, the range of TNF-\(\alpha\) concentrations used to establish an inflammatory environment in NP cells was based on our workgroup's previous works [15] and does not reflect the tissue concentrations of TNF-\(\alpha\) found in humans, which is reported to be around 5.9-25.9 pg/mL depending on the duration of complaints [80]. As this is a preclinical experiment in 2D NP cells, the inflammatory environment shown in our in vitro model cannot exactly simulate the inflammatory situation in vivo. Future degenerative disc in vivo models are needed here to translate these preclinical findings. Notably, we focused on changes in gene expression levels, and these changes might not reflect the changes in protein levels. Our ongoing studies involving different tRAS modulators, such as other AGTR1 inhibitors, will be conducted using expanded methodological techniques in order to visualize and quantify the tRAS components in NP cells and quantify protein concentrations of important inflammatory and tissue remodeling markers released by the cells. This expansion of methodological techniques is highly warranted to examine the pathways leading to the current results.