Abstract
Lipopolysaccharide (LPS), as the core pathogenic component of Gram negative bacterial cell walls, is one of the strongest inducers of innate immune response activation in the body. The LPS inflammatory model is currently one of the most commonly used experimental tools for studying infectious inflammation, sepsis, acute lung injury, and screening anti-inflammatory drugs. According to different experimental levels, LPS inflammation models can be divided into two categories: in vivo animal models and in vitro cell models. This article systematically explains the construction principle of LPS inflammation model, and provides a detailed introduction to systemic inflammation models, acute lung injury models, and in vitro cellular inflammation models based on macrophages, epithelial cells, etc. in commonly used animals such as mice and rats. The article summarizes the identification index system of the model (including behavioral, biochemical indicators, cytokines, histopathology, etc.), and analyzes the main factors affecting the stability of the model. This article aims to provide a systematic and actionable technical reference for establishing LPS inflammation models in the field of inflammation related research.
**Keywords * *: Lipopolysaccharide; Inflammatory model; Sepsis; Acute lung injury; RAW264.7 cells; Model identification
1. Introduction
Lipopolysaccharide is the main structural component of the outer membrane of Gram negative bacteria, released during bacterial lysis or proliferation, and can be efficiently recognized by the host immune system. LPS activates the MyD88 dependent and independent signaling pathways by binding to Toll like receptor 4 (TLR4), activating key transcription factors such as NF - κ B and MAPK, inducing the cascade release of a large number of inflammatory mediators such as tumor necrosis factor - α (TNF - α), interleukin-6 (IL-6), IL-1 β, and nitric oxide (NO), ultimately leading to local or systemic inflammatory responses.
The establishment of LPS inflammation model began in the 1980s, and after decades of development, a complete model system has been formed from molecular, cellular to whole animal levels. This model has outstanding advantages such as low modeling cost, easy operation, high repeatability, and clear pathological characteristics, and is widely used in fields such as sepsis, acute respiratory distress syndrome (ARDS), inflammatory bowel disease, neuroinflammation, and drug anti-inflammatory activity evaluation. However, the establishment of the LPS model involves multiple variable parameters such as strain source, dosage, administration route, animal strain, and detection time point. Improper selection may lead to experimental result bias or even model failure. Therefore, it is crucial for the system to master the standardized establishment method of LPS inflammation model.
2、 Overview of the molecular mechanism of LPS induced inflammation
LPS first binds to the LPS binding protein (LBP) in serum, forming a complex that presents LPS to the CD14 molecule, and then interacts with the TLR4/MD-2 receptor complex. After dimerization of the intracellular TIR domain of TLR4, the linker proteins MyD88 and Mal (TIRAP) are recruited to initiate the MyD88 dependent pathway. This pathway activates the I κ B kinase (IKK) complex and MAPK cascade through the IRAK family kinases, TRAF6, and TAK1. IKK phosphorylates and degrades I κ B α, releasing NF - κ B dimers into the nucleus and initiating transcription of pro-inflammatory genes such as TNF - α and IL-6. Meanwhile, the MAPK pathway (p38, JNK, ERK) activates the AP-1 transcription factor, synergistically enhancing the inflammatory response.
In addition, TLR4 can activate the MyD88 independent pathway through TRIF and TRAM adaptor proteins, leading to IRF3 activation and the production of type I interferon (IFN - α/β), participating in antiviral immunity and the pathological process of advanced sepsis. Understanding this signal network is the theoretical basis for optimizing LPS inflammation models and screening targeted drugs.
3、 Establishment of LPS induced inflammatory animal model in vivo
1. Selection of experimental animals
**Mice are the most commonly used LPS inflammatory model animals, mainly C57BL/6J and BALB/c strains. C57BL/6J mice are more sensitive to LPS induced endotoxin shock, while BALB/c mice are relatively less sensitive. **Rats (SD or Wistar) are also commonly used in LPS models due to their large size, which facilitates multiple blood collection and surgical procedures. **Rabbits and guinea pigs still have applications in certain specific studies, but they are no longer mainstream. Generally speaking, male animals are more commonly chosen to avoid interference with the inflammatory response caused by the estrogen cycle. The animal age is usually 6-8 weeks (mice weigh 18-22 g, rats weigh 200-250 g).
2. Model of systemic inflammation (endotoxemia)
This model simulates pus caused by gram-negative bacterial infection
Toxic syndrome/endotoxin shock is the most classic LPS in vivo model.
**Administration route and dosage * *:
-Intraperitoneal injection (i.p.): The most commonly used. Low dose (0.5-5 mg/kg) causes mild inflammatory response and is suitable for anti-inflammatory efficacy evaluation; High doses (10-20 mg/kg) can induce severe sepsis with a high mortality rate (50%~80% within 24 hours).
-Tail vein injection (i.v.): It has the fastest onset of action and strong inflammatory response, but requires high operational skills. The commonly used dose for mice is 5-15 mg/kg.
-Subcutaneous or intramuscular injection: slow absorption, mild inflammatory response, less commonly used in acute models.
**Typical pathological process: Serum TNF - α reaches its peak 1-2 hours after LPS injection, followed by an increase in IL-6 and IL-1 β; Changes in body temperature (first decreasing and then increasing), reduced activity, vertical hair, curling up, and other symptoms occur within 6-12 hours; Multiple organ damage (liver, kidney, lung) and a peak mortality rate may occur within 12-24 hours.
**Attention * *: The source and batch of LPS have a significant impact on the model. It is recommended to use LPS derived from Escherichia coli O111: B4 or O55: B5, and conduct preliminary experiments to determine the LD50.
3. Acute lung injury (ALI) model
LPS administration via airway can establish a localized pulmonary inflammation model and simulate acute respiratory distress syndrome.
**Establishment method * *:
-* * Intratracheal instillation * *: After anesthesia in mice, fix them supine and slowly inject LPS solution (20-100 μ L) into the mouth or tracheostomy tube using a microsyringe, with a dose of 1-5 mg/kg (or 10-100 μ g per mouse). After infusion, rotate the animal upright to evenly distribute LPS in both lungs.
-Nasal instillation: No surgery required, easy to operate. After mild anesthesia of mice, LPS solution (50-100 μ g/40 μ L) was dropped into both nostrils in portions and naturally inhaled. The lung inflammation formed by this method is slightly milder but more uniform.
**Model evaluation: After 6-24 hours of modeling, there was an increase in lung wet dry weight ratio, a significant increase in total protein and neutrophil count in bronchoalveolar lavage fluid (BALF), and an increase in levels of inflammatory factors (TNF - α, IL-6, MIP-2). HE staining of lung tissue shows thickening of alveolar septa, infiltration of inflammatory cells, formation of transparent membrane, and focal bleeding.
4. Other LPS induced specific inflammatory models
-Neuroinflammatory model: Central inflammatory response can be induced by intraventricular injection (i.c.v.) or stereotactic injection of LPS (1-10 μ g/animal) into the hippocampus or substantia nigra, which is used for research on Parkinson's disease and Alzheimer's disease.
-Arthritis Model: Intraarticular injection of LPS (10-30 μ g/joint) can induce a single arthritis model, resulting in joint swelling, synovial hyperplasia, and inflammatory cell infiltration.
-Enteritis model: LPS enema (1-5 mg/animal) can establish an acute intestinal inflammation model, or be used in combination with DSS to enhance enteritis phenotype.
4、 Establishment of LPS induced in vitro cellular inflammation model
1. Commonly used cell lines
LPS can directly act on various immune and non immune cells, inducing inflammatory responses. The most commonly used cell lines include:
-RAW264.7 (Mouse Macrophages): Highly sensitive to LPS, it is the preferred cell model for screening anti-inflammatory drugs.
-THP-1 (human monocytes): It needs to be differentiated by PMA induction before use, which is closer to the human response.
-BV-2 (mouse microglia): used for neuroinflammation research.
-A549 (human lung epithelial cells): used for studying the mechanism of lung inflammation.
-HUVEC (human umbilical vein endothelial cells): used for vascular inflammation models.
2. Key parameters for model establishment
**LPS concentration * *: Different cell types have significant differences in sensitivity to LPS. General recommendation scope:
-RAW264.7: 0.1~1 μ g/mL (commonly 1 μ g/mL)
-THP-1 (differentiated): 0.1~1 μ g/mL
- BV-2: 0.1~1 μg/mL
-A549: 1-10 μ g/mL (low sensitivity)
**Processing time * *: depends on the detection indicators. MRNA levels typically peak 2-6 hours after stimulation, protein secretion (TNF - α, IL-6) increases within 6-24 hours, and NO accumulates significantly after 12-24 hours. Suggest conducting time gradients (2, 4, 6, 12, 24 hours) for pre experiments.
**Cell state * *: Use logarithmic growth phase cells with moderate plate density (such as 1-2 × 10 ⁴ cells/well in a 96 well plate). It is recommended to replace fresh serum-free or low serum culture medium before LPS treatment to reduce the impact of serum components on LPS activity.
3. Common Problems and Optimization
-* * Decreased LPS activity * *: LPS stock solution should be packaged and stored at -20 ℃ to avoid repeated freezing and thawing. Pre ultrasonic treatment or vortex oscillation can restore its aggregation state and enhance its activity.
-Cytotoxicity: High concentrations of LPS (>10 μ g/mL) have direct toxicity to certain cells. It is recommended to set up CCK-8 or MTT cell viability assays simultaneously to ensure that the model conditions do not cause excessive cell death.
-Cross contamination of endotoxins: Culture vessels and reagents must be ensured to be free of endotoxins, otherwise it may cause an increase in background inflammatory reactions.
5、 Identification and evaluation indicators of models
1. In vivo model identification indicators
**Behavior and General State: Observing animal activity, vertical hair, curling, diarrhea, eye and nose secretions, etc. A clinical scoring system (0-4 points) can be designed to quantify the severity of the condition.
**Changes in body temperature: Using rectal temperature measurement method, there may be a decrease in body temperature (characteristic of endotoxin shock) in the early stage (1-2 hours) after LPS injection, followed by a rebound or sustained hypothermia (adverse prognostic indicator).
**Serum inflammatory factors * *: ELISA method for detecting TNF - α, IL-6, IL-1 β. Typical peak time: TNF - α (1-2 h), IL-6 (2-6 h). QRT PCR can also be used to detect the mRNA of inflammatory factors in organs such as the liver, lungs, and spleen.
**Organ damage markers * *: ALT/AST (liver injury), BUN/CRE (kidney injury), lung wet dry weight ratio, BALF total protein and cell count.
**Histopathology: Important organs such as the heart, liver, lungs, and kidneys undergo HE staining after fixation, embedding, and sectioning. Use a semi quantitative scoring system to evaluate changes in inflammatory cell infiltration, necrosis, edema, etc.
**Survival curve * *: suitable for mortality observation in high-dose LPS modeling, usually observed for 72 hours or 7 days.
2. In vitro model identification indicators
**Cell viability * *: CK-8 or MTT assay, ensuring viability ≥ 80% after LPS treatment.
**Inflammatory cytokine secretion * *: ELISA detection of TNF - α, IL-6, IL-1 β levels in the supernatant.
**NO generation * *: Griess method for detecting nitrite content.
**Signal pathway protein * *: Western blot detection of I κ B α degradation, p65, and p38/JNK/ERK phosphorylation; Immunofluorescence observation of p65 nuclear translocation.
**Inflammatory gene expression * *: qRT PCR detection of Tnf, Il6, Il1b, Nos2, Cox2 and other genes.
6、 Key factors affecting model stability
1. Source and quality of LPS
There were significant differences in LPS activity among different serotypes (O111: B4, O55: B5, O127: B8) and different extraction methods (phenol extraction, ultrafiltration). The same brand and batch of LPS should be used for the same experiment, and the batch number should be recorded.
2. Animal species and gender
C57BL/6 mice are more sensitive to LPS than BALB/c. The inflammatory response of female mice to LPS is relatively mild, which may be related to the immunomodulatory effect of estrogen. It is recommended to choose a single gender (usually male) to reduce variation.
3. Preparation and storage of LPS
LPS powder should be stored in the dark, dry, and at -20 ℃. When preparing, use sterile and pyrogen free PBS or physiological saline. Separate small tubes (e.g. 100 μ L/tube) to avoid repeated freezing and thawing. Thaw at room temperature before use and mix thoroughly.
4. Environmental factors
The temperature, humidity, circadian rhythm, and feed composition of animal feeding environments can all affect the intensity of inflammatory reactions. It is recommended to adaptively feed for at least one week before modeling, and the experiment should be conducted at a fixed time period (such as 9-11am).
7、 Application and limitations of the model
The LPS inflammatory model has been widely used in:
-* * Anti inflammatory drug screening * *: Evaluate the in vitro and in vivo anti-inflammatory activity of natural products, synthetic compounds, and biologics.
-* * Study on the mechanism of sepsis * *: Exploring the molecular mechanisms of immune paralysis, metabolic disorders, and multiple organ failure.
-* * Treatment strategy for acute lung injury * *: Evaluate the protective effects of stem cells, extracellular vesicles, gas molecules (H ₂ S, CO), etc.
-Neuroinflammation and Neuroprotection: Investigating the Role of Microglia Activation in Neurodegenerative Diseases.
However, the LPS model also has limitations. LPS induced inflammation is centered around "endotoxin shock" and differs from complex sepsis caused by live bacterial infections (involving bacterial dissemination, adaptive immune response, bacterial translocation, etc.). Therefore, the LPS model is more suitable for studying intrinsic immune driven inflammatory mechanisms, and is not applicable to chronic inflammation models that require adaptive immune involvement, such as CIA and EAE. In addition, different species have varying sensitivities to LPS (humans have much higher tolerance to LPS than mice), and conclusions based on mouse models should be cautiously extrapolated to clinical settings.
8、 Conclusion
The lipopolysaccharide inflammation model has become an indispensable experimental tool for studying inflammation mechanisms and discovering anti-inflammatory drugs due to its simple operation, controllable cost, clear pathological characteristics, and good human relevance. Whether it is an in vivo animal model or an in vitro cell model, establishing a standardized and reproducible modeling process, as well as a multidimensional and objective identification index system, is the key to obtaining reliable research data. With the continuous deepening of understanding of the LPS/TLR4 signaling pathway and the rise of new technologies such as organoids and organ chips, LPS inflammation models will further develop towards biomimetic, high-throughput, and clinical translation, providing stronger support for the research and treatment of inflammatory diseases.
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