Establishment of a lipopolysaccharide-induced inflammation model of human fetal colon cells

Inflammatory bowel disease (IBD) is a global health problem and there are few cell models for IBD at present. To culture a human fetal colon (FHC) cell line in vitro and establish an FHC cell inflammation model that meets the requirements for high expression of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). FHC cells were cultured with various concentrations of Escherichia coli lipopolysaccharide (LPS) in appropriate media for 0.5, 1, 2, 4, 8, 16 and 24 h to stimulate an inflammatory reaction. The viability of FHC cells was detected by a Cell Counting Kit-8 (CCK-8) assay. The transcriptional levels and protein expression changes of IL-6 and TNF-α in FHC cells were detected by Quantitative Real‑Time Polymerase Chain Reaction (qRT-PCR) and Enzyme‑Linked Immunosorbent Assay (ELISA), respectively. Appropriate stimulation conditions were selected (i.e., LPS concentration and treatment time), based on changes in cell survival rate, and IL-6 and TNF-α expression levels. An LPS concentration higher than 100 µg/mL or a treatment time longer than 24 h resulted in morphological changes and decreased cell survival. By contrast, expression levels of IL-6 and TNF-α significantly increased within 24 h when LPS concentration lower than 100 µg/mL and peaked at 2 h, whilst maintaining cell morphology and viability in FHC cells. The treatment of FHC cells with 100 µg/mL LPS within 24 h was optimal in terms of stimulating IL-6 and TNF-α expression.


Introduction
Condition of the digestive system Inflammatory bowel disease (IBD) is a chronic, non-specific inflammatory. Ulcerative colitis and Crohn's disease are the two most common types of IBD [1]. It has been reported that abnormal immune responses caused by heredity and environmental factors are major risk factors for the development of IBD [2]. In patients with IBD, a chronic inflammatory response of the intestinal tract can increase the risk of cancer development in the gastrointestinal system, including colon cancer, small intestinal sodium sulfate (DSS) are currently common cell and animal models for IBD research. The animal DSS model is wellestablished, with clearly defined stimulation conditions. However, for the cell inflammation model, a wide range of different stimulation conditions have been used among studies, along with a variety of cell types, including NCM460 [13,14], HT29 [15,16], and Caco2 [15] cells. Only a few studies have reported the use of human fetal colon (FHC) cells in recent years, among them, some researchers used proinflammatory cytokines to establish an FHC cell inflammation model [17], and others used DSS [18]. In our study, we induced an inflammatory response in FHC cells using LPS, and cell viability was determined by the CCK-8 method. The transcriptional levels of IL-6 and TNF-α and the protein expression changes in FHC cells were detected by qRT-PCR and ELISA, respectively. Based on these findings, we selected appropriate stimulation conditions (i.e., LPS concentration and treatment time), to establish a reliable inflammation model with high expression of IL-6 and TNF-α in FHC cells.

CCK-8 assay
FHC cells were digested, centrifuged, and resuspended, then inoculated into each well (5000 cells/well) of a 96-well plate. The plate was incubated for 1 h at 37 °C in 5% CO 2 . Following cell adhesion, LPS was added at different concentrations for different incubation times, then 10 µL of CCK-8 (BOSTER #AR1160, Wuhan, China) solution was added into each well and incubated for 1 h (37 °C, 5% CO 2 ). A microtiter plate reader (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the absorbance of the plate at 450 nm, and the OD 450 value was recorded.

Quantitative Real-time PCR and primers (qRT-PCR)
Total RNA was extracted from FHC cells using TRIzol (Takara #9109, Dalian, China) according to the manufacturer's protocol. Total RNA was reverse transcribed using a PrimeScript™ RT reagent kit with gDNA Eraser (Takara #RR047A). qRT-PCR was performed in triplicate using 2.5 to 10 ng of cDNA and TB Green® Premix Ex Taq™ II (Takara #RR820A) in a total volume of 10 µL on a CFX Connect™ Thermal Cycler (BIO-RAD, BR005222, Singapore). The relative quantities (D cycle threshold values) were obtained by normalization to the GAPDH gene, depending on the experiment. The thermal cycle: initial denaturation at 95˚C for 30 s, denaturation at 95˚C for 5 s, annealing at 60˚C for 30 s for a total of 40 cycles, and then at 65˚C for 5 s and at 95˚C for 5 s. The following primers were used:

Enzyme-Linked Immunosorbent Assay (ELISA)
The cell supernatant was centrifuged at 3000 rpm at room temperature for 30 min. Then, the supernatant was transferred to a fresh tube and the IL-6 and TNF-α levels were measured using a Human IL-6 (Jiubang #QZ-20,469, Fujian, China) ELISA kit and a Human TNF-α ELISA kit (Jiubang #QZ-20,789) according to the manufacturer's instructions.

Statistical analysis
One-way ANOVA was used to compare differences between multiple groups, Tukey's post hoc test followed for multiple comparisons. All data are shown as the mean ± standard error of the mean and each experiment was performed in triplicate in this study. Statistical analyses were performed using GraphPad Prism 8 software for analysis of variance with P < 0.05 considered statistically significant.

Effects of different concentrations of LPS on the morphology and viability of FHC cells
To explore the effect of LPS concentration on the viability and morphology of FHC cells, we treated FHC cells with different concentrations of LPS for 24 h. The results showed that when the LPS concentration was less than 31.25 µg/ mL, the number of viable cells and the cellular morphology were consistent with those in the control group (Fig. 1A-F).
When the concentration reached 156.25 µg/mL or higher, cell condition decreased and cell morphology changed, with the cells becoming rounder and smaller ( Fig. 1G and H). The results of the CCK-8 assay confirmed that when the concentration of LPS reached 156.25 µg/mL, cell viability was significantly decreased ( Fig. 2A).
To further explore the effects of LPS concentration and stimulation time on FHC cells, we cultured cells under different conditions, i.e., different LPS concentrations and different stimulation times. The results of the CCK-8 assay revealed that the viability of FHC cells was significantly decreased with longer incubation times of 36 and 48 h and viability was further decreased with increased LPS concentration ( Fig. 2D and E). Higher levels of viability were detected when FHC cells were cultured for 12 and 24 h with an LPS concentration of 100 µg/mL or less, when the concentration of LPS exceeded 100 µg/mL, the viability of FHC cells was significantly decreased ( Fig. 2B and C). This suggested that LPS concentrations greater than 100 µg/mL and incubation times greater than 24 h decrease the viability of FHC cells, which was consistent with the results in Fig. 1.

Effects of LPS on the mRNA expression of IL-6 and TNF-α
We next investigated the transcriptional effects of different LPS concentrations on FHC cell-associated inflammatory factors over a range of treatment times that did not affect the cell state. We cultured FHC cells with different concentrations of LPS for different times. QRT-PCR analysis revealed that the mRNA expression levels of IL-6 and TNF-α increased with the increase in LPS concentration over 24 h of culture, this increase was not significant at LPS concentrations of 16 µg/mL or lower, but when the LPS concentration was 64 µg/mL or higher, the mRNA expression levels of IL-6 and TNF-α were significantly increased (Figs. 3A-G and 4A-G). As we previously found that the viability of FHC cells begins to decrease significantly at LPS concentrations above 100 µg/mL, we added 100 µg/ mL LPS alone and incubated for different times. The results revealed that at an LPS concentration of 100 µg/mL, the mRNA expression levels of IL-6 and TNF-α significantly increased (> 3-4 times, respectively) within 24 h, with the expression levels peaking at 1 h after LPS stimulation (Figs. 3H and 4H).

Effect of LPS on the expression of IL-6 and TNF-α
We also determined the expression levels of IL-6 and TNF-α in the supernatants of FHC cells, cultured under the condi-  than 24 h, cell morphology was maintained and the number of viable cells remained high (i.e., inhibition rate < 10%). It was preliminarily determined that an LPS concentration of no more than 100 µg/mL was safe for modeling.
Inflammatory factors IL-6 and TNF-α can be used as an indicator to determine the severity of IBD [24,25]. Thus IL-6 and TNF-α were selected as markers to measure the success of the inflammatory model. Our results showed that when the concentration of LPS was 100 µg/mL and the treatment time was within 24 h, the mRNA and protein expression levels of IL-6 and TNF-α were significantly higher than those of the control group. However, the difference in mRNA expression was more pronounced than the corresponding difference in protein expression, which we propose may be related to mutual regulation of these inflammatory factors at the protein level. We also found that the peak of IL-6 and TNF-α expression in LPS-stimulated FHC cells was delayed by 1 h at the protein level relative to the mRNA level, which we propose may be related to the time required for protein translation and post-transcriptional regulation.
As an inflammatory cell model, we only evaluated the changes of inflammatory factors IL-6 and TNF-α, but it was Discussion LPS is a key cytotoxic factor causing inflammation [19], and it can be applied to induce and establish a variety of injury models [20,21]. To date, few studies have reported the use of LPS to stimulate inflammation in FHC cells. Furthermore, the LPS concentration used in previous studies was much lower than the concentration recommended in the current study [22,23]. A low LPS concentration may result in insignificant changes in the expression levels of inflammatory factors related to the inflammatory model, thus leading to inconclusive data. This may explain the poor application of this type of inflammatory model. Other common cell types used in inflammation models to date include a range of cancer cells, such as HT29 and Caco2. The only human normal colon cells that have been used for modeling are NCM460 and FHC cells. Of these two cell types, only FHC cells are available in the ATCC, so FHC cells were used to establish the cell inflammation model.
In this study, we first determined the optimal LPS concentration and incubation time for good cell growth without significant cell damage. We found that at an LPS concentration of 100 µg/mL or less and a culture time of no more , and 800 µg/mL for 12, 24 and 48 h. One-way ANOVA was used and Tukey's post hoc test followed. *P < 0.05 vs. control group, **P < 0.01 vs. control group, ***P < 0.001 vs. control group, ****P < 0.0001 vs. control group other hand, FHC cells can be co-cultured with immune cells to more closely mimic the in vivo environment of IBD.
In conclusion, we have successfully established a cell inflammation model by inducing FHC cells with LPS and determined that stimulation of FHC cells with 100 µg/mL LPS within 24 h could establish a reliable inflammation model in FHC cells of high expression of IL-6 and TNF-α, also, the expression levels of IL-6 and TNF-α peak at 2 h, which is of great reference value for the experiment of acute inflammation requiring high expression of IL-6 and TNF-α.
clear that IL-17, IL-10 and IL-1β also played a role. We were unable to produce the above factors at the detection level, possibly due to the lack of interaction between the various cells in our culture system. However, as classical markers of cellular inflammation, the significant increases of IL-6 and TNF-α provide sufficient evidence for the successful establishment of cellular inflammation models. Our further experiment envisaged that, on the one hand, cytokines secreted by immune cells could stimulate FHC cells; On the Acknowledgements Not applicable.
Author Contribution LL and ZM designed the study and performed the experiments, KY and YS collected the data, KY and SL analyzed the data, KY and CL prepared the manuscript. All authors read and ap- (I-P) Expression level of TNF-α was determined by ELISA. One-way ANOVA was used and Tukey's post hoc test followed. *P < 0.05 vs. control group, **P < 0.01 vs. control group, ***P < 0.001 vs. control group, ****P < 0.0001 vs. control group manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
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