Transcriptional analysis of the intestinal cells
To gain a comprehensive overview of
the transcriptional responses to basal fluid, we performed RNA-seq
analysis of intestinal epithelial cells in the chip. Differentially
expressed genes were identified to elucidate the transcriptomic
differences between the presence and absence of fluid. Based on the
biological repeated correlation test of the samples, a clear distinction
could be observed between the perfused and static groups, while within
each group, independent reproducibility was confirmed (Fig.
4B) . By applying the criteria of an absolute value of log fold change
> 1 and a p-value < 0.05, we identified 151
up-regulated genes and 304 down-regulated genes in the perfused group
compared to the static condition (Fig. 4A) .
The biological processes associated with these genes were further
characterized using GO and KEGG pathway enrichment analysis. Using the
GO database, we classified the differentially expressed genes into
biological process, molecular function, and cell component categories.
Among all the enriched terms, the biological process classification was
the most prominent and of primary interest. Fig. 4C presents
the top 20 terms in descending order of p-value. Notably, in the context
of our system, several up-regulated genes of cell under fluid flow were
associated with cellular activities, including “regulation of
epithelial cell proliferation”, “negative regulation of endothelial
cell apoptotic process”, and “cellular response to dexamethasone
stimulus”. Additionally, terms related to material metabolism, such as
”negative regulation of cholesterol storage”, ”linoleic acid metabolic
process”, and ”glutathione derivative biosynthetic process”, indicated
that fluid stimulation impacts the metabolic function of cells.
Interestingly, in the enrichment analysis of down-regulated genes, we
identified the terms “cellular response to fluid shear stress” and
“cellular response to mechanical stimulus”. Furthermore, terms related
to cell adhesion, such as “cell surface” and ”anchored component of
membrane”, were also present. Whether this is a negative feedback
regulation of cells in the gut-on-a-chip system we constructed, it still
needs a further exploration.
Furthermore, the KEGG enrichment analysis (Fig. 4D) revealed
that the presence of fluid in the culture enhances several pathways
associated with drug metabolism compared to static culture. These
pathways include “Metabolism of xenobiotics by cytochrome P450”,
“Drug metabolism - other enzymes”, and “Drug metabolism - cytochrome
P450”. Moreover, the enrichment of up-regulated genes in pathways such
as “Fat digestion and absorption” and “Metabolic pathways” indicates
an improvement in the metabolic function of cells in the presence of
fluid. These findings underscore
the ability of our platform to achieve better physiological function
simply by providing fluid with a rocker, as compared to regular static
cultivation.
Assessment of drug bioavailability after exposure to