Abstract
Lipopolysaccharide (LPS) is the main component of the outer membrane of Gram negative bacterial cells, with potent immune activation properties. It can stimulate various host cells such as monocytes, macrophages, endothelial cells, and epithelial cells, trigger intracellular signaling pathways, induce the production and release of a large number of inflammatory factors, and thus trigger inflammatory reactions. The LPS induced cellular inflammation model is widely used in the study of inflammatory disease mechanisms and the screening of anti-inflammatory drugs. This article systematically reviews the construction principles, commonly used cell lines, experimental parameter optimization strategies, identification and evaluation methods, and their applications in biomedical research of LPS in vitro inflammation models. It also explores the limitations and future development directions of this model, in order to provide legal references for researchers in related fields.
**Keywords * *: Lipopolysaccharide; In vitro cell model; Inflammation; Macrophages; RAW264.7 cells
1. Introduction
Inflammation is a complex and delicate defensive physiological response of the body to infection, tissue damage, or harmful stimuli, aimed at clearing pathogens, removing damaged tissues, and initiating repair processes. However, when the inflammatory response continues to be unbalanced or overactivated, it may evolve into a pathological state and participate in the occurrence and development of a variety of serious diseases, including septic shock, rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, chronic obstructive pulmonary disease and neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. In this context, establishing a reliable, controllable, and ethical inflammation research model has become a core prerequisite for elucidating inflammation mechanisms and developing anti-inflammatory drugs.
Lipopolysaccharide, as a unique pathological related molecular pattern, is recognized as a classic inducer of inflammatory response and plays an irreplaceable role in the construction of inflammatory models. Compared to animal level overall models, in vitro cell models have outstanding advantages such as easy operation, controllable cost, uniform conditions, high-throughput applicability, and ease of molecular mechanism research. LPS can directly act on various cell types cultured in vitro, by activating the Toll like receptor 4 (TLR4) - mediated signaling pathway, simulating key links in the inflammatory response in vivo.
In recent years, significant progress has been made in the optimization of experimental protocols, expansion of cell types, and multidimensional evaluation of LPS induced in vitro cellular inflammation models. This article will systematically review the molecular basis of model establishment, parameter optimization, commonly used cell lines, identification methods, and applications, providing reference for related research.
2、 Molecular basis of LPS induced inflammation
The inflammatory induction effect of LPS mainly stems from its specific recognition mechanism with the host's innate immune system. LPS activates complex and precise intracellular signaling by binding to TLR4 receptors and their co receptors (including CD14 and MD-2) on the cell membrane, ultimately triggering the expression of inflammation related genes.
Specifically, when LPS binds to the TLR4/MD-2 complex, the intracellular TIR domain of TLR4 undergoes dimerization, thereby recruiting the adaptor proteins MyD88 and Mal and initiating the MyD88 dependent signaling pathway. This pathway activates transcription factors NF - κ B and AP-1 through a series of kinase cascade reactions, promoting rapid expression of pro-inflammatory cytokines such as IL-1 β, TNF - α, IL-6, and IL-8. In addition, TLR4 can activate the MyD88 independent signaling pathway through the TRIF adaptor protein, activate the IRF3 transcription factor, and induce the production of type I interferon. The synergistic effect of two signaling pathways ensures a rapid and comprehensive response of the body to gram-negative bacterial infections. However, the excessive or sustained activation of this signaling system is also an important molecular basis for the occurrence of inflammation related diseases.
3、 Construction and optimization of in vitro cell models
1. Commonly used cell lines
At present, multiple cell lines have been successfully used for the construction of LPS induced in vitro inflammation models. **RAW264.7 * * cells are a mouse macrophage line that has become one of the most widely used cell models due to its sensitivity to LPS stimulation, strong proliferation ability, and ease of cultivation. After LPS stimulation, RAW264.7 cells can secrete a large amount of inflammatory mediators such as TNF - α, IL-6, NO, which have been used for anti-inflammatory drug screening and inflammation mechanism research.
**THP-1 * * cells are a human monocytic cell line that can differentiate into macrophage like cells after PMA induction, and are suitable for research on human inflammatory response and immune regulation. After LPS stimulation of THP-1 cells, the level of IL-6 in the supernatant significantly increased from (15.2 ± 4.8) pg/mL in the control group to (285.4 ± 32.7) pg/mL.
In addition, BV-2 microglia are widely used in neuroinflammation research, and when stimulated with 1 μ g/mL LPS, they exhibit significant secretion of inflammatory factors and activation of inflammasomes. **A549 * * Human lung epithelial cells are commonly used in research on lung injury and chronic obstructive pulmonary disease. **Caco-2 * * cells are used as intestinal epithelial barrier models for the study of intestinal inflammation. **HAECs * * human aortic endothelial cells showed decreased cell viability and increased inflammatory index after treatment with 100 μ g/mL LPS for 24 hours, which can be used for vascular inflammation research.
In fibroblast research, rabbit synovial fibroblast FLS can successfully establish an inflammatory model after 3 hours of stimulation with 1 μ g/mL LPS. WI-38 human embryonic lung fibroblasts were used for LPS induced fibroblast inflammatory response and differentiation studies.
In addition, with the development of cell modeling technology, some more complex and novel models are gradually being applied. For example, researchers have preliminarily constructed a mouse small intestine organoid culture system. After treating small intestine organoids with LPS at different times and concentrations, IL-10 levels were reduced, successfully inducing an in vitro intestinal inflammation injury model. The co culture system of Raw264.7 macrophages and Caco-2 cells was also used to simulate the inflammatory microenvironment of ulcerative colitis.
2. Source, dose, and treatment time of LPS
The source of LPS has a significant impact on the intensity of inflammatory response. The biological activity of LPS derived from different bacterial species varies - studies have shown that LPS from different sources, different administration methods, and different doses can all affect its effects. For example, LPS from gut commensal bacteria may have immunomodulatory rather than purely pro-inflammatory activity. In vitro cell models, the most commonly used source of LPS is the Escherichia coli O111: B4 strain, which is widely used due to its stable biological activity and small inter batch differences.
In the establishment of inflammation models, it is crucial to determine the appropriate LPS dosage and treatment time. **A dose that is too low may not be sufficient to induce a significant inflammatory response, while a dose that is too high may lead to excessive decrease in cell viability or even cell death, affecting subsequent testing.
-In RAW264.7 macrophages, the commonly used LPS concentration range is 0.1-10 μ g/mL.
-In A549 cells, LPS induced cell viability decreased, while IL-1 β, IL-6, and TNF - α secretion increased.
-In THP-1 cells, stimulation with 100 ng/mL LPS for 24 hours can cause a significant increase in IL-6.
-In the HAECs human aortic endothelial cell model, cell viability even showed a brief increase trend within 6 hours of LPS treatment; However, as the concentration of LPS increased to 50-100 μ g/mL for 24 hours, the cell viability significantly decreased, with a highly significant difference compared to the normal control group (P< 0.01). >
-In * * rabbit synovial fibroblast FLS * *, 1 μ g/mL LPS stimulation for 3 hours can successfully establish an inflammatory model.
**The optimization of processing time * * cannot be ignored. After LPS stimulation, the expression of inflammatory factors shows typical dynamic characteristics, and the peak time of different inflammatory factors varies. Therefore, researchers need to choose an appropriate processing time based on the target factors being detected.
3. Key parameters and precautions for model establishment
The successful construction of an LPS in vitro inflammatory model requires attention to multiple key parameters. Firstly, the cell state has a significant impact on the experimental results, and cells in logarithmic growth phase should be used for the experiment to ensure cell viability ≥ 95%. Secondly, the preparation and storage of LPS are particularly crucial. LPS powder is prepared as a 1 mg/mL stock solution in sterile PBS or culture medium, usually packaged and stored at -20 ℃. Repeated freezing and thawing should be avoided because LPS is more active in the form of endotoxin aggregates, and repeated freezing and thawing may damage its supramolecular structure, leading to inter batch differences. At the same time, the same batch of LPS should be used in the same experiment to ensure comparability of results. In addition, avoiding local high concentrations is an important detail in experimental procedures. When adding LPS to cell wells, it should be slowly added along the well wall and gently mixed. Local high concentrations may cause acute toxicity to cells. Finally, it is recommended to conduct a pre experiment to explore the gradient of LPS dosage and treatment time before the formal experiment, in order to determine the optimal conditions that can induce significant inflammatory response without causing excessive cell death. The principles are usually based on adjusting the dosage, shortening the exposure time, and finding conditions through pre experiments that can moderately increase inflammatory factors within an acceptable range of cell viability.
4、 Identification and evaluation methods for models
1. Cell viability testing
Cell viability testing is an important indicator to measure whether LPS treatment causes cell damage within an acceptable range. The CCK-8 method is currently the most commonly used method for detecting cell viability. Its principle is based on the reduction of WST-8 to orange yellow formamide by live cell mitochondrial dehydrogenase, and the amount of product generated is proportional to the number of live cells. Usually, after LPS treatment for 12 or 24 hours, about 10 μ L of CCK-8 reagent is added to each well, and incubation is continued for 1-4 hours before reading the absorbance value at a wavelength of 450 nm. To ensure successful model construction, the cell viability after LPS treatment should usually not be lower than 70%~80%, otherwise the increase in inflammatory factors may be partially due to non-specific release caused by cell death. For example, a trend of increasing and then decreasing cell viability was observed in HAECs cells treated with different concentrations of LPS. Therefore, it is necessary to select appropriate treatment conditions based on the dynamic changes in cell viability.
2. Detection of inflammatory cytokine expression
The expression level of inflammatory factors is a core indicator for evaluating the success or failure of LPS induced inflammation models. **The ELISA method can quantitatively detect the concentration of inflammatory cytokine proteins secreted in the supernatant of cell culture. After LPS stimulation, the cell supernatant was collected and the secretion levels of various inflammatory factors such as TNF - α, IL-6, IL-1 β, IL-8, etc. were detected by a double antibody sandwich ELISA method. **The qRT PCR method can detect the transcription levels of inflammatory cytokine genes in cells, usually using GAPDH or β - actin as internal reference genes, and performing relative quantitative analysis using the Δ Ct method.
In LPS induced cell models, the classic pattern of elevated inflammatory factors includes significant upregulation of TNF - α, IL-6, and IL-1 β. **The detection of nitric oxide (NO) * * is also an important evaluation indicator for macrophage inflammation models, and the Griess method is usually used to indirectly reflect the production of NO by detecting the nitrite content in cell culture supernatant.
3. Analysis of signaling pathway proteins
The activation of inflammatory signaling pathways is a core molecular event in the action of LPS, therefore the detection of signaling pathway proteins is of great value for understanding the mechanism of inflammation and evaluating intervention effects. **Western blot can detect the expression and phosphorylation levels of key proteins in the TLR4/NF - κ B pathway in cell lysate, such as the phosphorylation status of I κ B α, p65, p38, JNK, ERK, etc. Research has found that the activation of the NF - κ B signaling pathway after LPS stimulation is manifested by the degradation of I κ B α, phosphorylation of p65, and nuclear translocation. **Immunofluorescence assay can be used to observe the nuclear translocation process of NF - κ B p65- after LPS stimulation, p65 translocates from the cytoplasm to the nucleus, which is a visual evidence of NF - κ B pathway activation. In addition, the activation of NLRP3 inflammasome has become an important research hotspot in LPS induced inflammation models in recent years, which can be evaluated by detecting indicators such as caspase-1 cleavage, IL-1 β, and IL-18 mature secretion.
4. Observation of cellular morphology
LPS stimulation can induce significant changes in cell morphology and is a fast and intuitive method for model validation. In macrophages, LPS stimulation can induce an activated morphological transformation of cells from circular to irregularly shaped and pseudopodded, resulting in a more spreading morphology and enhanced phagocytic activity. In BV-2 microglia, a transition in cell morphology towards an activated state can also be observed after LPS stimulation. Morphological observation is usually combined with inverted microscopy, and the successful establishment of an inflammatory model can be preliminarily judged based on changes in cell morphology.
5、 Application and Prospect
The LPS induced in vitro inflammation model has broad application value in biomedical research. In the screening of anti-inflammatory drugs, researchers used the secretion level of inflammatory factors in cell supernatant as an indicator to optimize the LPS induction concentration, time, and positive drug dexamethasone incubation time, and investigate the anti-inflammatory activity of candidate compounds such as isoquinoline alkaloids. In * * mechanism research * *, this model can be used to explore the molecular pathways of LPS induced inflammatory response. For example, studies in porcine alveolar macrophages have found that the Notch signaling pathway can interact with the TLR signaling pathway to synergistically regulate the production of inflammatory factors and the activation of macrophages. In addition, this model can also be used to evaluate the inhibitory ability and immune regulatory effect of biomaterials, medical dressings, and other materials on inflammatory responses.
With the deepening of research, LPS in vitro inflammation models are also constantly developing towards more complex and biomimetic directions. The LPS inflammation model based on organoids, multiple cell co culture systems, and organ chip models on microfluidic chips will further enhance the ability of in vitro models to simulate physiological conditions in vivo. In addition, the differences in biological activity of LPS from different sources - some "excellent" LPS even exhibit immunomodulatory rather than pro-inflammatory effects - provide new dimensions for model design and drug evaluation, suggesting that researchers should choose LPS sources and construction strategies reasonably according to research purposes. The development of high-sensitivity detection technologies such as ultra sensitive ELISA and multi cytokine detection will also make model evaluation more refined and multidimensional. It can be foreseen that the LPS induced in vitro inflammation model will remain an indispensable basic tool in the field of inflammation research, and will continue to evolve and improve with technological innovation.
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