Low doses of lipopolysaccharide cause chronic inflammation in mice with chronic lung injury: specific roles for AMPK α2 knockout

doi:10.21203/rs.3.rs-2611325/v1

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Low doses of lipopolysaccharide cause chronic inflammation in mice with chronic lung injury: specific roles for AMPK α2 knockout

https://doi.org/10.21203/rs.3.rs-2611325/v1

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Background

Chronic inflammation leads to the activation of inflammatory signaling pathways, cause a long-term vicious cycle of inflammation. It exacerbates tissue and cellular damage in the body, especially in the lungs. Long-term treatment of Lipopolysaccharide (LPS) can cause this systemic chronic inflammation. It was recently proposed that 5’-AMP-activated kinase (AMPK), as an energy sensor, which regulates metabolic pathways and the response to cell stress. AMPK consists of three subunits containing α, β and γ subunits. Rare reports have been made regarding the role of AMPK α2 as a catalytic subunit of AMPK in lung injury caused by LPS.

Methods

Experimental validation was performed using mouse lung sections. HE staining was used to detect pathological and microstructural changes. Western blot, immunofluorescence, and enzyme-linked immunosorbent assay were used to evaluate protein expression.

Results

The structural alterations of the lung tissue in the AMPK α2 knockout group were noticeably better than those in the LPS group. IL-1, IL-6, IL-18, and TNF-α were all significantly lowered as well. Additionally, after AMPK α2 knockout, M-CSF, a marker of macrophage differentiation, was remarkable decreased. The relative levels of M2 type macrophage were significantly increased.

Conclusion

The results showed that AMPK α2 knockout reduces chronic inflammatory damage in the lung caused by LPS. This effect may be achieved by promoting macrophage differentiation into M2 type.

AMPK a2

Lipopolysaccharide

Inflammation

Chronic lung injury

Chronic inflammation is a state of persistent infection in the body stimulated by the long-term presence of low concentrations of inflammatory factors.¹ Normally, during chronic inflammation, the body continues to secrete small amounts of inflammatory cytokines and inflammatory mediators, which promotes the activation, secretion and accumulation of inflammatory cells such as neutrophils, eosinophils and monocytes. This leads to the activation of inflammatory signaling pathways, resulting in a prolonged vicious cycle of inflammation that exacerbates tissue and cellular damage in the body.^2,3

Lipopolysaccharide (LPS) is a chemical component of the outer wall of Gram-negative bacteria and is commonly used by researchers as a model for acute and chronic inflammation due to its stability and convenience.⁴ Palikov VA developed a model of the chronic lung inflammation by repeated administration of LPS in a dose of 0.4 mg/kg male rats.⁵ On day 22 of the study, have relative monocytopenia and reduced mean concentration of hemoglobin in the erythrocyte and the mean platelet volume in comparison with the control animals (saline). Acute lung injury modeled by either inhalation or systemic (intravenous and intraperitoneal) administration LPS within a short period. It showed damage to various organs in the 24h.^6,7 Different concentrations and administration methods of LPS induced different inflammatory responses. We speculate that it is possible that different concentrations of LPS caused different degrees of lung damage.

Adenosine 5‘-monophosphate (AMP) -activated protein kinase is a conserved energy sensor that plays a key role in maintaining cellular energy homeostasis. AMPK regulates cellular energy status and prevents cellular stress by sensing energy homeostasis when energy in the cell decreases. AMPK consists of three subunits containing α, β and γ subunits.⁸ Of these, the N-terminal catalytic core site in α subunit serves as a marker for AMPK activation.⁹ As an energy sensor, AMPK can be activated by increased intracellular AMP levels, disease stress or energy abnormalities.¹⁰ Once activated, AMPK acts through downstream proteins, including ACC, to stimulate metabolic catabolic pathways such as glucose uptake and fatty acid oxidation, restoring cellular energy levels and reducing fatty acid, cholesterol and protein synthesis.¹¹

Recently, AMPK has been reported to play an important role in the regulation of inflammation.¹² Research shows that, AMPK can regulate inflammation by indirectly regulates NF-kB (nuclear factor-k-gene binding) pathway through downstream proteins such as SIRT1 (Silent mating type information regulation 2 homolog- 1), FoxO3a (Fork-head box o3), p53 (Tumor Protein p53) and PGC-1α (peroxisome proliferators-activated receptor γ co-activator l alpha).^13–17 It has been demonstrated that in the lung injury induced by smoke inhalation, the AMPK inhibitor Compound C can inhibit macrophage function.¹⁸ While macrophages are one of the important inflammatory cells that regulate the inflammatory response and can promote inflammation.¹⁹ This result suggests that the AMPK signaling pathway is associated with immune cell activation, and specifically may be related to the energy requirements of the cells upon activation.²⁰ At the same time, systemic knockout of AMPK α2 may inhibit the metabolic demands and energy replenishment of body cells and reduce LPS-induced signaling cascades, cytokine production and immune cell activation. In addition, AMPK inhibitors have been shown to exert significant protective effects in LPS-induced acute hepatitis.²¹ What about the role of AMPK α2 in LPS-induced chronic inflammatory lung injury?

In order to investigate the possible role of AMPK α2 in LPS-induced chronic inflammatory lung injury, the present experiments were conducted to select AMPK α2 systemic knockout C57 mice for the intraperitoneal injection of LPS-induced chronic inflammation model, with reference to the membrane building method previously reported in the literature. The goal of this study was to investigate the potential role of AMPK α2 in LPS-induced chronic inflammatory lung injury and to provide new experimental information for the prevention and treatment of chronic inflammatory lung injury. This was done by observing the function of AMPK α2 to the damage on lung tissue and the level of inflammatory response.

Animals

Wild type and AMPK α2 KO male mice (C57BL/6 background) were studied at 8–12 wk of age. Animals were housed under controlled temperature and lighting with free access to water and standard mice chow diet. AMPK α2 KO male mice were purchased from Beijing Vitalstar Biotechnology Co.,Ltd. The SPF animals were kept in the Hubei Key Laboratory of Diabetes and Angiopathy, Medicine Research Institute, Xianning Medical College, Hubei University of Science and Technology. All experiments were conducted under protocols approved by the Experimental Animal Management Regulations in Hubei Province.

Lps-induced Chronic Inflammatory Lung Injury Mice Model

Mice were divided into four groups randomly: Group 1: Wild type group (n = 10), Group 2: LPS group(0.4 mg/kg) (n = 25), Group 3: AMPK α2 KO group (n = 25), Group 4: AMPK α2 KO + LPS group༈0.4 mg/kg) (n = 25). LPS (0.4 mg/kg, from Escherichia coli O111: B4, Sigma-Aldrich, USA) dissolved in 0.9% saline. Intraperitoneal injection once every two days, three injections in total, the concentration of the injection is 40 µg/ mL.

Mouse Genotyping Kit

Following the instructions of the One Step Mouse Genotyping Kit, the first step is DNA extraction. Depending on the number of samples to be lysed, the appropriate amount of 1 × lysis solution is prepared. Added 200 µl of 1 × lysate to the tissue to be lysed, vortexed and incubated in a water bath at 55°C for 20 min. After incubation, the sample was inactivated by heating at 95℃ or in a boiling water bath for 5 min. The lysate was whirled and mixed thoroughly, then centrifuged at 12,000 rpm (13,400 × g) for 5 min and the supernatant was removed for PCR (Polymerase Chain Reaction) reaction. The amplification products were directly detected by agarose gel electrophoresis.

Hematoxylin And Eosin (He) Staining

Mice lung tissues in 4% paraformaldehyde fixed liquid soaked overnight. Gradient ethanol solution was used for dehydration the next day. Soaking the specimens in xylene until it was amber-transparent. The transparent specimens were placed in melted paraffin wax at 65◦C for 2 hours. After the paraffin was completely embedded, cooled and solidified for the wax block. First of all, the tissue were cut into 4 ms slices then placed the slices in hot water and ironed them. Finally, seal these slices for use.

Paraffin sections were baked in 65° oven for 30 minutes and soaked in xylene for dewaxing, paraffin sections were rehydrated in various levels of alcohol in turn, stained with hematoxylin for 4 minutes, washed with water for 7 minutes, dehydrated in alcohol, then stained with eosin for 10 seconds, washed with alcohol to remove excess color and dehydrated again, transparent with xylene related solution, and finally sealed with neutral resin.

Enzyme-linked Immunosor Bent Assay (Elisa)

Levels of IL-6, IL-1β, TNF-α, IL-18 and M-CSF, in lung homogenates were measured by corresponding ELISA kits (Abcam).

Immunofluorescence Staining

After deparaffinization, rehydration and antigen retrieval, the sections of lung tissue were incubated with primary antibodies against CD86 (1:100; A2353, ABclonal, Wuhan, China) and CD206 (1:2000; 60143-1-lg, Proteintech group, Wuhan, China) at 4 ◦C overnight, and then incubated with fluorescent-labeled secondary antibodies for 30 min. After being counterstained with DAPI, the sections were imaged using a fluorescent microscope.

Western-blot

The lung tissue of mice was taken, the lysate was fully centrifuged, and the protein concentration was determined. The primary antibodies used to target proteins was as follows: OPA1 (1:1000; A9833, ABclonal, Wuhan, China); DRP1 (1:1000; A2586, ABclonal, Wuhan, China); IL18 (1:1000; A16737, ABclonal, Wuhan, China); AMPKα (1:1000; 2532s, CST, Shanghai, China); p-AMPKα (1:1000; 2535s, CST, Shanghai, China); AMPK α2 (1:1000; 32082, Upstate, USA); PGC-1α (1:1000; GR181813-6, Abcam, Wuhan, China).

Statistical Analysis

Analyze and process images using Image J and image-Pro-plus software to obtain data. Group differences were determined by the One-way analysis or t test of variance using Graph Pad Prism version 8.0.2 software. P values of < 0.05 were considered statistically significant.

P-AMPK expression are increased in LPS-induced chronic inflammation

AMPK, as an energy sensor, significantly activated at a low dose LPS-treatment. The flow chart shows how we make the animal model (Figure. 1A). Western-blot shows that p-AMPK gene expression was significantly higher in LPS-treated mice with compared to the normal group (Figure. 1B). Statistical plots shows that the total AMPK protein nothing has been changed (Figure. 1C), but p-AMPK / AMPK (Figure. 1D) and p-AMPK / β-actin relative change (Figure. 1E) are significantly different.

Ampk α2 Knockout Attenuates Lps-induced Chronic Lung Injury

The chronic inflammation caused by LPS usually causes damage to various organs, especially the lung. Electrophoresis shows that AMPK α2 knockout success (Figure. 2A). The flow chart shows how we make the animal model (Figure. 2B). Western-blot (Figure. 2C) and statistical plots (Figure. 2C) show that AMPK α2 knockout success. Lung tissue sections showed significant differences in the lung structure between normal control and LPS group. A series of significant histological abnormality appears in the LPS group than normal group, including alveolar wall thickening, congestion, neutrophil infiltration and alveolar collapse. This damage phenomenon significant relieve after AMPK α2 knockout than LPS group (Figure. 2D).

Ampk α2 Knockout Reduces Lps-induced Production Of Pro-inflammatory Factors

The chronic inflammation caused by LPS usually causes elevated levels of many inflammatory factors. The ELISA results showed that LPS group caused a significant increase than normal group in TNF-α, IL-6 and IL-1β levels. Whereas AMPK α2 knockout treatment significantly inhibited the increase than normal group in TNF-α, IL-6 and IL-1β levels (Figure. 3A-C). Consistent with this result, western blot and statistical plots results showed that LPS group appeared a significant increase than normal group in IL-18 levels, while AMPK α2 knockout treatment significantly inhibited the increase than LPS group in IL-18 levels (Figure. 3D-E).

Ampk α2 Knockout Restored Lps-induced Macrophage Changes

We also used enzyme-linked immunosorbent assay to detect the level of in the lung tissue supernatant of mice in each group. The results showed that LPS group induced a significant increase than normal group in M-CSF levels, whereas AMPK α2 knockout treatment significantly inhibited the increase in M-CSF levels (Figure. 4A). These results suggest that AMPK α2 knockout has a mitigating effect on inflammation in chronic lung injury, and that this mitigating effect may be related to the inhibition of macrophage differentiation and proliferation. Next, we examined the ratio of macrophage type M1 to type M2. Immunofluorescence staining and statistical plots show that in LPS group, the ratio of CD82 to CD206 significantly higher than normal group. However, this ratio had significantly decrease after AMPK α2 knockout (Figure. 4B, C).

No Change In The Expression Of Mitochondria-related Proteins

We want to verify whether this phenomenon is related to mitochondria-related proteins. The results showed that LPS group induced nothing changed than normal group in mitochondria-related proteins expression, and nothing has changed after AMPK α2 knockout, Such as Drp1, OPA1, PGC1-α. This scenario suggests that this inflammatory lung injury may not be related to mitochondrial changes (Figure. 5).

Schematic Representation Of Ampk α2 Regulation Of Inflammation In Low Dose Lps-induced Lung Injury

Schematic diagram shows that AMPK α2 knockout promotes macrophage differentiation and reduces LPS-induced production of inflammatory factors (Figure. 6).

The lung is one of the target organs of endotoxin, and stimulation by continual low doses of LPS increases the permeability of lung tissue cell membranes, causing accumulation of edematous fluid between the alveolar lumen and the interstitial space, edema of lung tissue, congestion of the alveolar lumen, and infiltration and aggregation of inflammatory cells around the pulmonary vascular wall, triggering massive secretion of inflammatory factors and inflammatory mediators, ultimately leading to diffuse alveolar damage.³

The energy receptor AMPK is essential for intracellular energy homeostasis, and recent studies have also shown that AMPK is also involved in regulating a variety of high energy-consuming pathological processes such as inflammation and apoptosis.²⁰ We have previously found that AMPK can play an important role in the liver injury by promoting apoptosis.^21–23 It has been suggested that the downstream effects of AMPK activation may vary depending on the intensity of tissue stress and metabolic demand.²⁴ Recently, AMPK has been shown to regulate airway trans-epithelial ion transport, inflammation and oxidative stress. So we verified whether a defect in AMPK α2 could ameliorate LPS-induced lung injury due to chronic inflammation in rats.

In the present study, we noted that LPS stimulation caused a series of significant histological abnormalities in the lung tissue including structural damage, inflammatory cell infiltration and oxidative stress. These abnormalities were significantly alleviated by the intervention of AMPK α2 knockout. We identified AMPK α2 knockout as a protective factor in the LPS-induced chronic inflammatory state, where AMPK α2 is upregulated, and AMPK α2 knockout reduced LPS-induced inflammatory cell infiltration in lung tissue and decreased the expression of unregulated inflammatory factors such as IL-6, TNF-α, IL-18 and IL-1β, And AMPK α2 knockout significantly suppressed the overexpression of Macrophage colony-stimulating factor (M-CSF) due to LPS treatment. M-CSF, also known as Colony-stimulating factor 1 (CSF-1), is a spectrum-specific cells.M-CSF is a dimeric glycoprotein linked by interchain disulfide bonds which is mainly found in Bone marrow cavity macrophage cells and has a major role in their proliferation. It is important for macrophage differentiation and activity. So, we used immunofluorescence to detect any changes in macrophages. We found that, M2 macrophage relative content decreases after LPS treatment, whereas AMPK α2 knockout significantly rebound M2 macrophage relative content. M1 macrophages and M2 macrophages are closely related to inflammatory responses, among which M1 macrophages are mainly involved in pro-inflammatory responses and M2 macrophages are mainly involved in anti-inflammatory responses.²⁵ In our study, we speculate that AMPK α2 knockout may regulate macrophage polarization through macrophage colony-stimulating factor, thus playing a protective role in LPS-induced chronic lung inflammation.

Therefore, this study tentatively demonstrates that AMPK α2 is involved in the development of chronic inflammation, possibly by modulating the value-added differentiation of macrophages, altering the secretion levels of pro-inflammatory factors, and possibly also closely related to immune function, thereby modulating the lung injury due to chronic inflammation, and is expected to be a new drug target for chronic inflammatory lung injury.

The reason our results differ from others may be due to the fact that the model we used was a mild induction of LPS, resulting in a milder response. We speculate that the early stages of the disease, AMPK α2 knockout acts as a delaying agent for the onset of the inflammatory response. If the disease is advanced or the LPS dose is increased, this delay in the onset of the disease could turn into a worsening of the disease process.

In summary, in this study, we found that AMPK α2 deficiency in the lung tissue of LPS-induced chronic inflammatory mice. AMPK α2 knockout mice slowed down the pathological features of lung tissue injury and reduced inflammatory cell infiltration, as well as reduced the level of pro-inflammatory cytokine secretion and alleviated lung injury. Further studies are needed to identify the specific ways in which AMPK α2 regulates lung injury in this disease and to provide a new idea for the treatment of LPS-induced lung injury.

Competing interests

These authors declare that they have no competing interests.

Ethics approval and consent to participate

All experiments were approved by the Ethics Committee of Hubei University of Science and Technology(SYXK2018-0071). All methods were carried out in accordance with the relevant guidelines and regulations. This study was conducted in accordance with the arrival guidelines (https://arriveguidelines.org).

Consent for publication

Not applicable.

Availability of data and materials

The data used to support the findings of this study are available from the corresponding author upon request.

Funding

This work was supported by the National Natural Science Foundation of China (82270892), Development and utilization of experimental animal resources in Hubei Province (2021DFE025), Natural Science Foundation of Hubei Province (2022CFB287), Xianning City Science and Technology Plan Project (2022ZRKX052), School projects of Hubei University of Science and Technology (2021WG05, 2021TNB01).

Author contribution

Binbin Wanand Yuyang Ningperformed most of the experiments and analysed the data. Yue Zhang and Yuting Yuan revised the manuscript. Ninghua Wu participated in the discussion of the manuscript. Chao Liu and QingJie Chen participated in its design and coordination. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Author details

¹ School of Pharmacy, Xianning Medical College, Hubei University of Science and Technology, Xiannning, 437100, PR China

² Hubei Key Laboratory of Diabetes and Angiopathy, Medicine Research Institute, Xianning Medical College, Hubei University of Science and Technology, Xiannning, 437100, PR China

³School of Basic medical Sciences, Xianning Medical College, Hubei University of Science and Technology, Xiannning, 437100, PR China

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No competing interests reported.

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Version 1

posted

You are reading this latest preprint version

Low doses of lipopolysaccharide cause chronic inflammation in mice with chronic lung injury: specific roles for AMPK α2 knockout

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Introduction

Methods And Materials

Animals

Lps-induced Chronic Inflammatory Lung Injury Mice Model

Mouse Genotyping Kit

Hematoxylin And Eosin (He) Staining

Enzyme-linked Immunosor Bent Assay (Elisa)

Immunofluorescence Staining

Western-blot

Statistical Analysis

Result

P-AMPK expression are increased in LPS-induced chronic inflammation

Ampk α2 Knockout Attenuates Lps-induced Chronic Lung Injury

Ampk α2 Knockout Reduces Lps-induced Production Of Pro-inflammatory Factors

Ampk α2 Knockout Restored Lps-induced Macrophage Changes

No Change In The Expression Of Mitochondria-related Proteins

Schematic Representation Of Ampk α2 Regulation Of Inflammation In Low Dose Lps-induced Lung Injury

Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1